Compounds that enhance Atoh1 expression

ABSTRACT

This invention generally provides compounds, pharmaceutical compositions, and methods for their use, which include methods that result in increased expression in an Atoh1 gene (e.g., Hath1) in a biological cell. More specifically, the invention relates to the treatment of diseases and/or disorders that would benefit from increased Atoh1 expression, e.g., a hearing impairment or imbalance disorder associated with a loss of auditory hair cells, or a disorder associated with abnormal cellular proliferation.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.15/240,303, filed Aug. 18, 2016, which is a continuation of U.S. patentapplication Ser. No. 14/511,911, filed on Oct. 10, 2014, now U.S. Pat.No. 9,433,610, which is a continuation of U.S. patent application Ser.No. 13/454,702, filed Apr. 24, 2012, now U.S. Pat. No. 8,859,597, whichis a divisional of U.S. patent application Ser. No. 12/368,173, filedFeb. 9, 2009, no U.S. Pat. No. 8,188,131, which claims the benefit under35 USC § 119(e) of U.S. Provisional Patent Application Ser. No.61/027,032, filed on Feb. 7, 2008, the entire contents of each of theseprior filed applications is incorporated herein by reference.

TECHNICAL FIELD

This invention generally provides compounds, pharmaceuticalcompositions, and methods for their use, which include methods thatresult in increased expression in an Atoh1 gene (e.g., Hath1) in abiological cell. More specifically, the invention relates to thetreatment of diseases and/or disorders that would benefit from increasedAtoh1 expression.

BACKGROUND

One of the most common types of hearing loss is sensorineural deafnesswhich is caused by the loss of hair cells, sensory cells in the cochleathat are responsible for transduction of sound into an electricalsignal. The human inner ear contains only about 15,000 hair cells percochlea at birth, and, although these cells can be lost as a result ofvarious genetic or environmental factors, the lost or damaged cellscannot be replaced. However, overexpression of the transcription factor,Atoh1, can induce the differentiation of hair cells from epithelialcells in the sensory organ of the cochlea, the organ of Corti ((Zhengand Gao, Nat Neurosci 2000; 3:580-586; Kawamoto et al., J Neurosci 2003;23:4395-4400; Gubbels et al., Nature 2008; 455:537-541). Atoh-1expression also plays a role in driving other cells, e.g., intestinalcells, into a differentiated state (Aragaki et al., Biochem. Biophys.Res. Comm. 2008 April; 368(4):923-929), and overexpression of Atoh-1reduces proliferation of colon cancer cells (Leow et al., Ann NY AcadSci. 2005 November; 1059:174-83).

SUMMARY

The present invention features the compounds described herein, andcompositions containing them. For example, the present inventionfeatures a pharmaceutical composition including one or more compoundscapable of increasing Atoh1 expression in a cell, as disclosed herein,or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier. The compositions can be formulated foradministration to a patient. Thus, pharmaceutical compositions arewithin the present invention, as are concentrated stocks andcompositions suitable for application to cells or tissues maintained intissue culture, and methods of use of the compounds and compositions.

Where the composition is pharmaceutically acceptable (i.e., non-toxic),it can include a pharmaceutically acceptable carrier such as a buffer(e.g., a phosphate buffer), an amino acid, urea, an alcohol, ascorbicacid, a phospholipid, a polypeptide, EDTA, sodium chloride (e.g., normalsaline), a liposome, mannitol, sorbitol, water, glycerol, or acombination thereof. Preservatives and dyes may also be included. Insome embodiments, the composition is sterile.

The compounds described herein can be used to alter the characteristicsof a cell maintained in cell culture (e.g., in vitro), and the compoundsand/or the treated cells can be administered to a patient in need oftreatment. For example, a method of treating a patient can be carriedout by a method including the steps of (a) selecting a patient in needof treatment, and (b) administering to the patient a therapeuticallyeffective amount of a compound described herein (e.g., a compoundformulated for administration). The pharmaceutical composition can beadministered systemically (e.g., orally or parenterally). Morespecifically, the composition can be administered intravenously,intramuscularly, intraperitoneally, sublingually, rectally, vaginally,transdermally, subcutaneously, or by inhalation. When administeredorally, the composition can be formulated as a tablet (e.g., acompressed tablet), pill, syrup, suspension, emulsion, or capsule. Whenadministered parenterally, the composition can be formulated as alozenge, drop (e.g., ear drops), solution, enema, suppository, or spray.The compositions can also be administered using a catheter or pump.

The present compositions can also be administered locally (e.g., to theear or other site where cellular differentiation and/or Atoh1 expressionis desired). For administration to the ear, the pharmaceuticalcomposition can be administered by injection into the luminae of thecochlea, into the auditory nerve trunk in the internal auditory meatus,and/or into the scala tympani. More specifically, the pharmaceuticalcomposition can be administered by intratympanic injection, applicationto (e.g., injection into) the outer, middle, or inner ear, an injectionthrough the round window of the ear, or an injection through thecochlear capsule. The pharmaceutical composition can also beadministered to the patient (e.g., locally to the middle and/or innerear) using a catheter or pump.

The patient in need of treatment can have, or have a risk of developing,a hearing impairment or imbalance disorder associated with loss ofauditory hair cells. While the invention is not limited to compoundsthat work by any particular mechanism, the present compositions may beused where the treatment effectively increases the expression of anAtoh1 gene in cells in the patient's inner ear (or other target tissue(e.g., a tumor)) or effectively increases the number of cells in thepatient's inner ear that have characteristics of auditory hair cells.The auditory hair cells can be outer or inner auditory hair cells.

The patient in need of treatment can also have, or be at risk ofdeveloping, cancer. The cancer can be a gastrointestinal cancer (e.g.,cancer of the esophagus, gallbladder, liver, pancreas, stomach, smallintestine, large intestine, colon, or rectum).

The patient in need of treatment can also have, or be at risk ofdeveloping, cerebellar granule neuron deficiencies, joint disease,and/or osteoarthritis.

In one embodiment, the method of treating a patient who has a hearingimpairment or imbalance disorder can be carried out by a method thatincludes the steps of: (a) optionally selecting a patient in need oftreatment, (b) obtaining a population of cells capable ofdifferentiating into auditory hair cells, (c) contacting the populationof cells in vitro with an effective amount of one or more of thecompounds described herein for a time sufficient to increase the numbercells in the population that have characteristics of a differentiatedauditory hair cell, and (d) administering the population of cells, or asubset thereof (e.g., a subset of more highly differentiated cells), tothe patient's ear. The population of cells capable of differentiatinginto auditory hair cells can include stem cells, induced pluripotentstem (iPS) cells, progenitor cells, support cells, Deiters' cells,pillar cells, inner phalangeal cells, tectal cells, Hensen's cells, andgerm cells. The stem cells can be adult stem cells (e.g., stem cellsderived from the inner ear, bone marrow, mesenchyme, skin, fat, liver,muscle, or blood), embryonic stem cells, or stem cells obtained from aplacenta or umbilical cord. Like the stem cells, the progenitor cellscan be derived from the inner ear, bone marrow, mesenchyme, skin, fat,liver, muscle, or blood. Administering the population of cells can beaccomplished by (a) injecting the cells into the luminae of the cochlea,into the auditory nerve trunk in the internal auditory meatus, or intothe scala tympani or (b) implanting the cells within a cochlear implant.In any method where the patient is treated with cells, they may, inaddition, be treated with one or more of the present compounds, andvice-versa. The pharmaceutical compositions can be administeredsystemically or locally, as described above.

Other methods of the invention include methods of increasing the numberof cells with the characteristics of auditory hair cells in a populationof cells in vitro. These methods can be carried out by obtaining apopulation of cells capable of differentiating into auditory hair cells,contacting the population of cells in vitro (e.g., in cell culture) withan effective amount of one or more of the compounds described herein fora time sufficient to increase the number of cells with thecharacteristics of auditory hair cells in the population of cells. Thepopulation of cells capable of differentiating into hair cells includescells selected from the group consisting of stem cells, iPS cells, innerear stem cells, adult stem cells, bone marrow derived stem cells,embryonic stem cells, mesenchymal stem cells, skin stem cells, fatderived stem cells, progenitor cells, inner ear progenitor cells,support cells, Deiters' cells, pillar cells, inner phalangeal cells,tectal cells, Hensen's cells, and germ cells.

Also within the invention is the use of the compounds described hereinas a medicament, and in the manufacture of a medicament for thetreatment or prevention of a condition described herein. For example,the medicament can be used in a method for treating or preventinghearing loss or imbalance associated with hair cell loss, or a conditionassociated with unwanted cellular proliferation. Also within the presentinvention is the use of the described compounds in the treatment of acondition described herein, e.g., hearing loss or imbalance associatedwith hear cell loss, or a condition associated with unwanted cellularproliferation. The medicament can be in any form described herein, andcan be administered alone or in combination with another treatment oractive agent.

Also provided herein are kits (e.g., a kit comprising the pharmaceuticalcompositions described above with informational material or a kitcomprising a compound described herein and informational material). Thecells within the kits can be made by the methods described above, andany of the kits can include additional materials such as a devicesuitable for administration of the pharmaceutical composition or thepopulation of cells, e.g., a sterile flexible cannula that is adaptedfor insertion into the inner ear of a subject.

Further, the invention encompasses a cell or a population of cells madeby the methods described herein.

The present disclosure also includes using one or more of the compoundsdescribed herein as a medicament, e.g., that can be used for thetreatment of a hearing impairment or imbalance disorder associated witha loss of auditory hair cells and/or a condition associated withabnormal cellular proliferation.

The use of one or more of the compounds described herein for thetreatment of a hearing impairment or imbalance disorder associated witha loss of auditory hair cells and/or a disorder associated with abnormalcellular proliferation is also encompassed by the present disclosure.

Definitions

The term “abnormal proliferation” as used herein is defined as anyunwanted hyperproliferation of any type of cell, wherein said cell isnot under the constraints of normal cell cycle progression and whereinsaid proliferation can result in a tumor or any cancerous development.

As used herein, “treatment” means any manner in which one or more of thesymptoms of a disease or disorder are ameliorated or otherwisebeneficially altered. As used herein, amelioration of the symptoms of aparticular disorder refers to any lessening, whether permanent ortemporary, lasting or transient that can be attributed to or associatedwith treatment by the compositions and methods of the present invention.

The terms “effective amount” and “effective to treat,” as used herein,refer to an amount or a concentration of one or more compounds or apharmaceutical composition described herein utilized for a period oftime (including acute or chronic administration and periodic orcontinuous administration) that is effective within the context of itsadministration for causing an intended effect or physiological outcome.

Effective amounts of one or more compounds or a pharmaceuticalcomposition for use in the present invention include amounts thatpromote increased Atoh1 expression, promote complete or partialdifferentiation of one or more cells to treat a disease that wouldbenefit from increased Atoh1 expression, e.g., prevent or delay theonset, delay the progression, ameliorate the effects of, or generallyimprove the prognosis of a patient diagnosed with one or more diseasesthat would benefit from increased Atoh1 expression, e.g., one or more ofthe diseases described herein. For example, in the treatment of hearingimpairment, a compound which improves hearing to any degree or arrestsany symptom of hearing impairment would be therapeutically effective. Inthe treatment of abnormal proliferation of cells, a compound whichreduces proliferation would be therapeutically effective. In thetreatment of abnormal cell proliferation, a compound which reducesproliferation of the cells, reduces tumor size, reduces metastases,reduces proliferation of blood vessels to said cancer would betherapeutically effective, for example. A therapeutically effectiveamount of a compound is not required to cure a disease but will providea treatment for a disease.

The term “patient” is used throughout the specification to describe ananimal, human or non-human, to whom treatment according to the methodsof the present invention is provided. Veterinary and non-veterinaryapplications are contemplated. The term includes, but is not limited to,birds, reptiles, amphibians, and mammals, e.g., humans, other primates,pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters,cows, horses, cats, dogs, sheep and goats. Typical patients includehumans, farm animals, and domestic pets such as cats and dogs.

The term “halo” or “halogen” refers to any radical of fluorine,chlorine, bromine or iodine.

In general, and unless otherwise indicated, substituent (radical) prefixnames are derived from the parent hydride by either (i) replacing the“ane” in the parent hydride with the suffixes “yl,” “diyl,” “triyl,”“tetrayl,” etc.; or (ii) replacing the “e” in the parent hydride withthe suffixes “yl,” “diyl,” “triyl,” “tetrayl,” etc. (here the atom(s)with the free valence, when specified, is (are) given numbers as low asis consistent with any established numbering of the parent hydride).Accepted contracted names, e.g., adamantyl, naphthyl, anthryl,phenanthryl, furyl, pyridyl, isoquinolyl, quinolyl, and piperidyl, andtrivial names, e.g., vinyl, allyl, phenyl, and thienyl are also usedherein throughout. Conventional numbering/lettering systems are alsoadhered to for substituent numbering and the nomenclature of fused,bicyclic, tricyclic, polycyclic rings.

The term “alkyl” refers to a saturated hydrocarbon chain that may be astraight chain or branched chain, containing the indicated number ofcarbon atoms. For example, C₁-C₆ alkyl indicates that the group may havefrom 1 to 6 (inclusive) carbon atoms in it. Any atom can be optionallysubstituted, e.g., by one or more substitutents (e.g., such as thosedelineated in any definition of R^(a) described herein). Examples ofalkyl groups include without limitation methyl, ethyl, n-propyl,isopropyl, and tert-butyl.

The term “haloalkyl” refers to an alkyl group, in which at least onehydrogen atom is replaced by halo. In some embodiments, more than onehydrogen atom (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) arereplaced by halo. In these embodiments, the hydrogen atoms can each bereplaced by the same halogen (e.g., fluoro) or the hydrogen atoms can bereplaced by a combination of different halogens (e.g., fluoro andchloro). “Haloalkyl” also includes alkyl moieties in which all hydrogenshave been replaced by halo (sometimes referred to herein asperhaloalkyl, e.g., perfluoroalkyl, such as trifluoromethyl). Any atomcan be optionally substituted, e.g., by one or more substituents (e.g.,such as those delineated in any definition of R^(b) described herein).

The term “aralkyl” refers to an alkyl moiety in which an alkyl hydrogenatom is replaced by an aryl group. One of the carbons of the alkylmoiety serves as the point of attachment of the aralkyl group to anothermoiety. Any ring or chain atom can be optionally substituted e.g., byone or more substituents (e.g., such as those delineated in anydefinition of R^(c) described herein). Non-limiting examples of“aralkyl” include benzyl, 2-phenylethyl, and 3-phenylpropyl groups.

The term “heteroaralkyl” refers to an alkyl moiety in which an alkylhydrogen atom is replaced by a heteroaryl group. One of the carbons ofthe alkyl moiety serves as the point of attachment of the aralkyl groupto another moiety. Heteroaralkyl includes groups in which more than onehydrogen atom on an alkyl moiety has been replaced by a heteroarylgroup. Any ring or chain atom can be optionally substituted e.g., by oneor more substituents (e.g., such as those delineated in any definitionof R^(c) described herein). Heteroaralkyl can include, for example,2-pyridylethyl.

The terms “alkoxy” and “haloalkoxy” refer to —O-alkyl and —O-haloalkylradicals, respectively. The term “phenoxy” refers to an —O-phenylradical.

The term “heterocyclyl” refers to a fully saturated monocyclic,bicyclic, tricyclic or other polycyclic ring system having one or more(e.g., 1-4) heteroatom ring atoms independently selected from O, N, orS. The heteroatom or ring carbon is the point of attachment of theheterocyclyl substituent to another moiety. Any atom can be optionallysubstituted, e.g., by one or more substituents (e.g., such as thosedelineated in any definition of R^(c) described herein). Heterocyclylgroups can include, e.g., tetrahydrofuryl, tetrahydropyranyl, piperidyl(piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, andpyrrolidinyl.

The term “cycloalkyl” refers to a fully saturated monocyclic, bicyclic,tricyclic, or other polycyclic hydrocarbon groups. Any atom can beoptionally substituted, e.g., by one or more substituents (e.g., such asthose delineated in any definition of R^(c) described herein). A ringcarbon serves as the point of attachment of a cycloalkyl group toanother moiety. Cycloalkyl moieties can include, e.g., cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, andnorbornyl (bicycle[2.2.1]heptyl).

The term “cycloalkenyl” refers to partially unsaturated monocyclic,bicyclic, tricyclic, or other polycyclic hydrocarbon groups. A ringcarbon (e.g., saturated or unsaturated) is the point of attachment ofthe cycloalkenyl substituent. Any atom can be optionally substitutede.g., by one or more substituents (e.g., such as those delineated in anydefinition of R^(c) described herein). Cycloalkenyl moieties caninclude, e.g., cyclohexenyl, cyclohexadienyl, or norbornenyl.

The term “aryl” refers to an aromatic monocyclic or bicyclic hydrocarbonring system, wherein any ring atom can be optionally substituted, e.g.,by one or more substituents (e.g., such as those delineated in anydefinition of R^(d) described herein). Aryl moieties can include phenyland naphthyl.

The term “heteroaryl” refers to an aromatic monocyclic or bicyclichydrocarbon groups having one or more (e.g., 1-6) heteroatom ring atomsindependently selected from O, N, or S (and mono and dioxides thereof,e.g., N→O⁻, S(O), SO₂). Any atom can be optionally substituted, e.g., byone or more substituents (e.g., such as those delineated in anydefinition of R^(d) described herein). Heteroaryl groups can includepyridyl, thienyl, furyl (furanyl), imidazolyl, isoquinolyl, quinolyl andpyrrolyl.

The descriptor C(O) refers to a carbon atom that is doubly bonded to anoxygen atom. The term “oxo” refers to doubly bonded oxygen, i.e., ═O.

The term “substituent” refers to a group “substituted” on, e.g., analkyl, haloalkyl, cycloalkyl, aralkyl, heteroaralkyl, heterocyclyl,cycloalkenyl, aryl, or heteroaryl group at any atom of that group. Ingeneral, when a definition for a particular variable includes bothhydrogen and non-hydrogen (halo, alkyl, aryl, etc.) possibilities, theterm “substituent(s) other than hydrogen” refers collectively to thenon-hydrogen possibilities for that particular variable.

Descriptors such as “C₆-C₁₀ aryl which is optionally substituted withfrom 1-5 R^(d)” (and the like) is intended to include both anunsubstituted C₆-C₁₀ aryl group and a C₆-C₁₀ aryl group that issubstituted with from 1-5 R^(d). The use of a substituent (radical)prefix names such as alkyl without the modifier “optionally substituted”or “substituted” is understood to mean that the particular substituentis unsubstituted. However, the use of “haloalkyl” without the modifier“optionally substituted” or “substituted” is still understood to mean analkyl group, in which at least one hydrogen atom is replaced by halo.

For ease of exposition, it is also understood that where in thisspecification (including the claims), a group is defined by “as definedanywhere herein” (or the like), the definitions for that particulargroup include the first occurring and broadest generic definition aswell as any sub-generic and specific definitions delineated anywhere inthis specification.

This application is related to U.S. Provisional Application Ser. No.60/605,746, filed on Aug. 31, 2004, International Application No.PCT/US2005/030714, filed on Aug. 30, 2005, U.S. application Ser. No.10/989,649, filed on Nov. 15, 2004, U.S. application Ser. No.11/953,797, filed on Dec. 12, 2007, U.S. application Ser. No.12/187,543, filed on Aug. 7, 2008, U.S. Provisional Application Ser. No.60/859,041, filed on Nov. 15, 2006, International Application No.PCT/US2007/084654, filed on Nov. 14, 2007, U.S. application Ser. No.12/233,017, filed Sep. 18, 2008, and U.S. Provisional Application Ser.No. 60/859,041, filed Nov. 24, 2008, the entire contents of each ofwhich are incorporated herein by reference.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1H are generalized structures of phenolic compounds orderivatives thereof.

FIGS. 1I-1K are structures of specific phenolic compounds or derivativesthereof.

FIGS. 2A-2F are generalized structures of benzamide compounds or relatedcompounds.

FIGS. 2G-2I are structures of specific benzamide compounds or relatedcompounds.

FIGS. 3A-3X are generalized structures of compounds that include one ormore heterocyclic rings.

FIGS. 3Y-3ZZ are structures of specific compounds that include one ormore heterocyclic rings.

FIGS. 4A-4G are generalized structures of compounds that include one ormore phenyl rings.

FIGS. 4H-4I are structures of specific compounds that include one ormore phenyl rings.

FIGS. 5A-5E are generalized structures of compounds that include anamide group attached to a 5-membered heterocyclic ring system.

FIGS. 5F and 5G are structures of specific compounds that include anamide group attached to a 5-membered heterocyclic ring system.

FIGS. 6A-6O are generalized structures of compounds that include a5-membered heterocyclic ring system fused to another ring system.

FIGS. 6P-6V are structures of specific compounds that include a5-membered heterocyclic ring system fused to another ring system.

FIGS. 7A-7C are generalized structures of pyridine compounds, while FIG.7D is a generalized structure of pyrimidine compounds.

FIGS. 7E-7F are structures of specific pyridine or pyrimidine compounds.

FIGS. 8A and 8B are generalized structures of aniline compounds oraniline derivatives.

FIG. 8C are structures of specific structures of anilines or anilinederivatives.

FIGS. 9-116 are line graphs showing Math1 expression in HEK cells with astably expressed Luciferase gene controlled by a Math1 enhancer andminimal promoter (see Example 1). The compound numbers indicated in thegraphs correspond to the compound structures presented in FIG. 1 to FIG.8. Math1 activation was measured using the high throughput screeningmethods described in Example 1 and Example 2.

FIGS. 117A and 117B are graphs showing the results of experimentsperformed to optimize the high throughput screen.

FIGS. 118A and 118B are dot plots showing the a duplicate experimentperformed to optimize the high throughput screen.

FIG. 119 is a bar graph showing Math1 activation, as assessed using theMath1 Luciferase reporter assay described in Example 1 and Example 2, incells exposed to the indicated compounds. The structure of thesecompounds is presented in FIG. 1 to FIG. 8. Initial Atoh1 activationresults for these compounds can be found in FIGS. 9-114.

FIG. 120A is a bar graph showing Math 1 activation, as assessed usingthe Math1 Luciferase reporter assay described in Example 1 and Example2, in cells exposed to the indicated compounds. The structure of thesecompounds is presented in FIG. 1 to FIG. 8. Initial Math 1 activationresults for these compounds can be found in FIGS. 9-114.

FIG. 120B shows the structures of the compounds indicated in FIG. 117A.

FIG. 120C shows Atoh1 mRNA expression in cells exposed to the indicatedcompounds.

FIGS. 121A and 121B are photographs of untreated cells (A) and cellscontacted with compound (Cp) Cp.−0000540 (B). Cell populations thatstained positive for the hair cell specific markers Math1-GFP and myosin7a are indicated by arrows.

DETAILED DESCRIPTION

The present invention provides, inter alfa, compounds and methodsrelated to compounds and/or pharmaceutical compositions for treatingpatients for the conditions described herein. While the treatmentmethods are not limited to those in which particular underlying cellularevents occur, the present compounds and compositions may increase theexpression of an Atoh1 gene in a subject and/or a cell, thereby causingthe cell to differentiate, e.g., into an auditory hair cell.

Atoh-1

Atonal protein homologue 1 (Atoh1 or atonal) is a proneural gene thatencodes a basic helix-loop-helix (bHLH) domain-containing protein thatseems to play an important role in cell fate determination in thedevelopment of the Drosophila nervous system (Jarman et al., Cell,73:1307-1321, 1993). Atoh1 is evolutionarily conserved, with homologsidentified in Tribolium castenium (the red flour beetle), Fugu rubripes(puffer fish), chicken (Cath1), mouse (Math1), and human (Hath1)(Ben-Arie et al., Hum. Mol. Gene., 5:1207-1216, 1996). Each of thesehomologs contain a bHLH domain that is identical in length and have highsequence identity to the Atoh1 bHLH domain. For example, the Hath1 andMath1 genes are almost identical in length. These molecules also havehighly similar nucleotide sequences (86% identity) and highly similarbHLH amino acid sequences (89%). The bHLH domain of Cath1 is 97% and 95%identical to the bHLH domain of Hath1 and Math1, respectively. The bHLHof Cath1 is 67% identical to the Atoh1 bHLH domain. In contrast, thebHLH domains of other Drosophila encoded proteins share only 40-50%sequence identity.

Each of the mammalian Atoh1 homologs functions as a transcription factorthat activates E box (CANNTG (SEQ ID NO:1)) dependent transcription(Arie et al., Hum. Mol. Genet., 9:1207-1216, 1996; Akazawa et al., J.Biol. Chem., 270:8730-8738, 1995) and functions as a critical positiveregulator of cell fate determination in neural tissue and thegastrointestinal (GI) tract (Helms et al., Development, 125:919-928,1998; Isaka et al., Eur. J. Neurosci., 11:2582-2588, 1999; Ben-Arie etal., Development, 127:1039-1048, 2000).

The use of nucleic acids encoding the Atoh1 homologues described abovefor the treatment of deafness, osteoarthritis, and abnormal cellproliferation was described by Zoghbi et al., (U.S. Publication No.2004/0237127).

As used herein, “Atoh1” refers to any and all Atoh1-associated nucleicacid or protein sequences and includes any sequence that is orthologousor homologous to, or has significant sequence similarity to, an Atoh1nucleic acid or amino acid sequence, respectively, and thus the term“Atoh1” includes other mammalian homologues, e.g., human, mouse, rat,etc. The sequence can be present in any animal including mammals (e.g.,humans). Examples of Atoh1 nucleic acid and amino sequences include, butare not limited to Atoh1 (e.g., NM_001012432.1 and NP_001012434.1,respectively)(Pan troglodytes), Hath1 (e.g., NM_005172.1 andNP_005163.1)(Homo sapiens), Math1 (e.g., NM_007500.4 andNP_031526.1)(Mus musculus), Atoh1 (NM_001109238.1 and NP_001102708.1)(Rattus norvegicus); Atoh1 (XM_001102247.1 and XP_001102247.1)(Macacamulatta); Atoh1 (NM_001098099.1 and NP_001091568.1)(Bos taurus); Atoh1(XM_544986.2 and XP_544986.2)(Canis lupus familiaris); and Cath1 (e.g.,U61149.1 and AF467292.1)(Gallus gallus), as well as all other synonymsthat may be used to refer to this protein, e.g., atonal, atonal homolog1, Ath1, and helix-loop-helix protein Hath1. Furthermore, multiplehomologous or similar sequences can exist in an animal. See, e.g.,GeneID: 474 (Homo sapiens); GeneID: 11921 (Mus musculus); GeneID: 461380(Pan troglodytes); GeneID: 500156 (Rattus norvegicus); GeneID: 704893(Macaca mulatta); GeneID: 539158 (Bos taurus); and GeneID: 487864 (Canislupus familiaris).

Any sequence with significant sequence similarity (i.e., similaritygreater than 80%, e.g., is at least 85%, 90%, 95%, 99%, or more, acrossthe entire sequence) to the human Atoh1 sequence (found at Genbank Acc.Nos. NM_005172.1 and NP_005163.1) can be used in the present methods. Todetermine the percent identity of two nucleic acid sequences, thesequences are aligned for optimal comparison purposes (gaps areintroduced in one or both of a first and a second amino acid or nucleicacid sequence as required for optimal alignment, and non-homologoussequences can be disregarded for comparison purposes). The length of areference sequence aligned for comparison purposes is at least 80% (insome embodiments, about 85%, 90%, 95%, or 100% of the length of thereference sequence) is aligned. The nucleotides at correspondingnucleotide positions are then compared. When a position in the firstsequence is occupied by the same nucleotide as the correspondingposition in the second sequence, then the molecules are identical atthat position. The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two amino acidsequences can be determined using the Needleman and Wunsch ((1970) J.Mol. Biol. 48:444-453) algorithm which has been incorporated into theGAP program in the GCG software package, using a Blossum 62 scoringmatrix with a gap penalty of 12, a gap extend penalty of 4, and aframeshift gap penalty of 5.

Compounds

The present invention provides compounds that are capable of increasingAtoh1 expression in a cell. In some embodiments, the increase in Atoh1expression is a significant increase. In some embodiments, the increasein Atoh1 expression can be, e.g., between about 1-10% above baseline,11-20%, 21-30%, 31-40%, 41-50%, 51-60%, 61-70%, 71-80%, 81-90%, 91-100%,101-200%, 201-300%, 301-400%, 401-500%, 501-1000%, 1001-10000%,10001-100000% or more. Increases in Atoh1 can also be expressed as afold increase, e.g., wherein a increase of 100% is a 1-fold increase, anincrease of 1000% is a 10-fold increase and so forth. Alternatively orin addition, the increase in Atoh1 expression is sufficient to promotethe differentiation of a cell, e.g., of a non-auditory hair cell (i.e.,a cell other than an auditory hair cell, e.g., a progenitor or stemcell) to or towards an auditory hair cell.

The compounds that can be utilized in any of the methods describedherein are phenolic compounds (or their sulfur analogs, e.g., phenylthiols), or compounds that are derived from such compounds, such asphenyl ethers (or thioethers), e.g., straight chain or cyclic phenylethers. For example, such compounds can be generally represented bythose structures shown in FIGS. 1A-1H, and specifically exemplified inthose structures shown in FIGS. 1I-1K. Any phenolic compound (or sulfuranalog) can be in neutral or salt form, e.g., a lithium, sodium,potassium or calcium salt thereof.

Such phenolic compounds and derivatives (or their sulfur analogs) aredescribed by the structures of FIG. 1A, in which R₂, R₃, R₄, R₅ and R₆are each independently H, F, Cl, Br, I, OH, CN, NO₂, or a moiety thatincludes up to 16 carbon atoms and, optionally, one or more N, O, S or Fatoms; X is O (see FIG. 1B) or S (see FIG. 1C); and R₁ is H or a moietythat includes up to 16 carbon atoms and, optionally, one or more N, O, Sor F atoms. For example, the moiety that includes up to 16 carbon atomsand, optionally, one or more N, O, S or F atoms can be an alkoxy groupor a trifluoromethyl group.

Referring to FIG. 1D, in particular embodiments, R₁ and R₂ togetherdefine one or more ring systems that each include up to 16 carbon atomsand, optionally, one or more N, O, S or F atoms.

Referring to FIG. 1E, in particular embodiments, R₁ is H; that is, thecompounds are phenols.

Referring to FIGS. 1F-1H, in certain embodiments, the phenolicderivatives are cyclic ether derivatives. For example, the cyclic etherportion of the molecules can be made rigid by incorporating a carbonylgroup (see FIG. 1F) and/or a carbon-carbon double bond (see FIG. 1G,which contains both). In other embodiments, such cyclic etherderivatives can be made rigid by incorporating a second ring systemabout the cyclic ether system. In particular embodiments, the cyclicether derivatives are represented by the structure of FIG. 1F in whichR₈, R₉, R₁₀ and R₁₁ are each independently H, F, Cl, Br, I, OH, CN, NO₂,or a moiety that includes up to 16 carbon atoms and, optionally, one ormore N, O, S or F atoms. In other particular embodiments, the cyclicether derivatives are represented by the structure of FIG. 1G, in whichR₁₂ and R₁₃ are each independently H, F, Cl, Br, I, OH, or a moiety thatincludes up to 16 carbon atoms and, optionally, one or more N, O, S or Fatom. In other particular embodiments, the cyclic ether derivatives arerepresented by the structure of FIG. 1H, in which R₁₄ and R₁₃ togetherdefine one or more ring systems that each include up to 16 carbon atomsand, optionally, one or more N, O, S or F atoms, R₁₆, R₁₇ and R₁₈ eachare each independently H, F, Cl, Br, I, OH, or a moiety that includes upto 16 carbon atoms and, optionally, one or more N, O, S or F atoms.

The present compounds are benzamide compounds and/or related compounds.Such compounds can be generally represented by those structures shown inFIGS. 2A-2F, and specifically exemplified in those structures shown inFIGS. 2G-2I. Any of the present benzamide or related compounds can be inneutral or salt form.

The present benzamide compounds and/or derivatives can be described bythe structure of FIG. 2A, in which R₂₀, R₂₁, R₂₂, R₂₃ and R₂₄ are eachindependently H, F, Cl, Br, I, OH, CN, NO₂, or a moiety that includes upto 16 carbon atoms and, optionally, one or more N, O, S or F atoms; andR₂₄ and R₂₆ are each independently H or a moiety that includes up to 16carbon atoms and, optionally, one or more N, O, S or F atoms.

In the compounds of FIG. 2A, R₂₄ and R₂₅ can together define one or morering systems that each include up to 16 carbon atoms and, optionally,one or more N, O, S or F atoms. Such compounds can be represented by thestructures of FIG. 2B. For example, such compounds can have thestructures shown in FIGS. 2C and 2D in which R₂₇, R₂₈, R₂₉, R₃₁ and R₃₂are each independently H, F, Cl, Br, I, OH, CN, NO₂, or a moiety thatincludes up to 16 carbon atoms and, optionally, one or more N, O, S or Fatoms; and R₂₆ is H or a moiety that includes up to 16 carbon atoms and,optionally, one or more N, O, S or F atoms.

Other benzamide-related compounds and derivatives are described by thestructures of FIGS. 2E and 2F, in which R₃₃, R₃₄, R₃₅, R₃₆ and R₃₇ areeach independently H, F, Cl, Br, I, OH, CN, NO₂, or a moiety thatincludes up to 16 carbon atoms and, optionally, one or more N, O, S or Fatoms; R₃₉, R₄₀, R₄₁ and R₄₂ are each independently H or a moiety thatincludes up to 16 carbon atoms and, optionally, one or more N, O, S or Fatoms.

The present compounds are, or can include, one or more heterocyclic ringsystems, such as a 3, 4, 5, 6, or 7-membered ring system that includesone more heteroatoms, such as O, S or N. For example, the one or morering systems can include 1, 2, 3, 4 or even 5 heteroatoms, such as O, Sor N. In many embodiments, the rings systems are aromatic. For example,such compounds can be generally represented by those structures shown inFIGS. 3A-3X, and specifically exemplified in those structures shown inFIGS. 3Y-3ZZ. Any described compound that is or that includes the one ormore ring system can be in neutral or salt form.

The compounds that are, or that include, one or more heterocyclic ringsystems are described by the structures of FIG. 3A, in which R₄₃ and R₄₄are each independently H, F, Cl, Br, I, OH, CN, NO₂, or a moiety thatincludes up to 16 carbon atoms and, optionally, one or more N, O, S or Fatoms; and X is O (FIG. 3B) or S (FIG. 3C).

Compounds that are or include one or more heterocyclic ring systems aredescribed by the structures of FIG. 3D, in which R₄₅, R₄₆ and R₄₈ areeach independently H, F, Cl, Br, I, OH, CN, NO₂, or a moiety thatincludes up to 16 carbon atoms and, optionally, one or more N, O, S or Fatoms; and R₄₇ and R₄₉ are each independently H, or a moiety thatincludes up to 16 carbon atoms and, optionally, one or more N, O, S or Fatoms.

Compounds that are or include one or more heterocyclic ring systems aredescribed by the structures of FIG. 3E, in which R₅₀, R₅₁, R₅₂ and R₅₃are each independently H, F, Cl, Br, I, OH, CN, NO₂, or a moiety thatincludes up to 16 carbon atoms and, optionally, one or more N, O, S or Fatoms; and X is O (FIG. 3F) or S (FIG. 3G).

Compounds that are or include one or more heterocyclic ring systems aredescribed by the structures of FIG. 3H, in which R₅₅, R₅₆ and R₅₇ areeach independently H, F, Cl, Br, I, OH, CN, NO₂, or a moiety thatincludes up to 16 carbon atoms and, optionally, one or more N, O, S or Fatoms; and R₅₄ is H or a moiety that includes up to 16 carbon atoms and,optionally, one or more N, O, S or F atoms. For example, in specificembodiments, R₅₄ and R₅₅ can together define one or more ring systemsthat each includes up to 16 carbon atoms and, optionally, one or more N,O, S or F atoms, as shown in FIG. 3I.

Compounds that are or include one or more heterocyclic ring systems aredescribed by the structures of FIG. 3J, in which R₅₇, R₅₉ and R₆₀ areeach independently H, F, Cl, Br, I, OH, CN, NO₂ or a moiety thatincludes up to 16 carbon atoms and, optionally, one or more N, O, S or Fatoms; and R₅₈ is H or a moiety that includes up to 16 carbon atoms and,optionally, one or more N, O, S or F atoms. For example, in specificembodiments, R₅₈ and R₅₉ can together define one or more ring systemsthat each includes up to 16 carbon atoms and, optionally, one or more N,O, S or F atoms, as shown in FIG. 3K. For example, in specificembodiments, R₅₇ and R₆₀ can together define one or more ring systemsthat each includes up to 16 carbon atoms and, optionally, one or more N,O, S or F atoms, as shown in FIG. 3L. For example, in specificembodiments, R₅₇ and R₆₀ and R₅₈ and R₅₉ can together each define one ormore ring systems that each includes up to 16 carbon atoms and,optionally, one or more N, O, S or F atoms, as shown in FIG. 3M.

Compounds that are or include one or more heterocyclic ring systems aredescribed by the structures of FIG. 3N, in which R₆₁, R₆₂, and R₆₄ areeach independently H, F, Cl, Br, I, OH, CN, NO₂, or a moiety thatincludes up to 16 carbon atoms and, optionally, one or more N, O, S or Fatoms; and R₆₃ is H or a moiety that includes up to 16 carbon atoms and,optionally, one or more N, O, S or F atoms. For example, in specificembodiments, R₆₁ and R₆₂ can together define one or more ring systemsthat each includes up to 16 carbon atoms and, optionally, one or more N,O, S or F atoms, as shown in FIG. 3O.

Compounds that are or include one or more heterocyclic ring systems aredescribed by the structures of FIG. 3P, in which R₆₅ and R₆₆ are eachindependently H, F, Cl, Br, I, OH, CN, NO₂, or a moiety that includes upto 16 carbon atoms and, optionally, one or more N, O, S or F atoms; andX is O (FIG. 3Q) or S (FIG. 3R).

Compounds that are or include one or more heterocyclic ring systems aredescribed by the structures of FIG. 3S, in which R₆₇, R₆₈, and R₆₉ areeach independently H, F, Cl, Br, I, OH, CN, NO₂ or a moiety thatincludes up to 16 carbon atoms and, optionally, one or more N, O, S or Fatoms; and X is O (FIG. 3T) or S (FIG. 3U).

Compounds that are or include one or more heterocyclic ring systems aredescribed by the structures of FIG. 3V, in which R₇₀, R₇₁ and R₇₂ areeach independently H, F, Cl, Br, I, OH, CN, NO₂ or a moiety thatincludes up to 16 carbon atoms and, optionally, one or more N, O, S or Fatoms; and X is O (FIG. 3W) or S (FIG. 3X).

The present compounds, which can be utilized in any method describedherein, are or include one or more phenyl rings, such as a fused phenylring system, e.g., one that is part of a flavonoid, coumarin or othersimilar system. For example, such compounds can be generally representedby those structures shown in FIGS. 4A-4G, and specifically exemplifiedin those structures shown in FIGS. 4H and 4I. Any described compoundthat is or that includes the one or more ring system can be in neutralor salt form.

Compounds that are or include one or more phenyl rings are described bythe structures of FIG. 4A, in which R₇₄, R₇₅, R₇₆, R₇₇, R₇₈ and R₇₉ areeach independently H, F, Cl, Br, I, OH, CN, NO₂ or a moiety thatincludes up to 16 carbon atoms and, optionally, one or more N, O, S or Fatoms. For example, in specific embodiments, R₇₆ and R₇₇ can togetherdefine one or more ring systems that each includes up to 16 carbon atomsand, optionally, one or more N, O, S or F atoms, as shown in FIG. 4B.For example, the compounds of FIG. 4B, can be described by the compoundsof FIGS. 4C and 4D in which R₈₁, R₈₂, R₈₃ and R₈₄ are each independentlyH, F, Cl, Br, I, OH, CN, NO₂ or a moiety that includes up to 16 carbonatoms and, optionally, one or more N, O, S or F atoms. For example, inother specific embodiments in which R₇₆ and R₇₇ together define one ormore ring systems, the compounds can be represented by those structuresof FIGS. 4E-4G, in which R₈₅, R₈₆, R₈₇ and R₈₈ are each independently H,F, Cl, Br, I, OH, CN, NO₂ or a moiety that includes up to 16 carbonatoms and, optionally, one or more N, O, S or F atoms; and R₈₉ is H or amoiety that includes up to 16 carbon atoms and, optionally, one or moreN, O, S or F atoms.

In some embodiments, the compounds that can be utilized in any methoddescribed herein include an amide group bonded to a 5-memberedheterocyclic ring system, such as one that that includes one moreheteroatoms, such as O, S or N. For example, the one or more ringsystems can include 1, 2, 3, 4 or even 5 heteroatoms, such as O, S or N.In many embodiments, the rings systems are aromatic. For example, suchcompounds can be generally represented by those structures shown inFIGS. 5A-5E, and specifically exemplified in those structures shown inFIGS. 5F-5G. Any described compound that is or that includes the one ormore ring system can be in neutral or salt form.

In certain embodiments, such compounds that include an amide groupbonded to a 5-membered heterocyclic ring system are described by thestructure of FIG. 5A, in which R₉₂, R₉₃ and R₉₄ are each independentlyH, F, Cl, Br, I, OH, CN, NO₂ or a moiety that includes up to 16 carbonatoms and, optionally, one or more N, O, S or F atoms; and R₉₀ and R₉₁are each independently H or a moiety that includes up to 16 carbon atomsand, optionally, one or more N, O, S or F atoms; and X is O (FIG. 5B) orS (FIG. 5C). For example, in some specific embodiments, R₉₀ and R₉₁ cantogether define one or more ring systems that includes up to 16 carbonatoms, optionally, substituted with one or more N, O, S or F atoms (seeFIG. 5D).

In other certain embodiments, such compounds that include an amide groupbonded to a 5-membered heterocyclic ring system are described by thestructure of FIG. 5E, in which R₉₇ and R₉₈ are each independently H, F,Cl, Br, I, OH, CN, NO₂ or a moiety that includes up to 16 carbon atomsand, optionally, one or more N, O, S or F atoms; and R₉₅, R₉₆ and R₉₉are each independently H or a moiety that includes up to 16 carbon atomsand, optionally, one or more N, O, S or F atoms.

In some embodiments, the compounds that can be utilized in any methoddescribed herein include a 5-membered heterocyclic ring system fused toone or more other ring systems, e.g., that defines one or more 4-, 5-,6-, 7- or 8-membered ring system. For example, such compounds can begenerally represented by those structures shown in FIGS. 6A-6O, andspecifically exemplified in those structures shown in FIGS. 6P-6V. Anydescribed compound that is or that includes the one or more ring systemcan be in neutral or salt form.

In some embodiments, the compounds that include a 5-memberedheterocyclic ring system fused to one or more other ring systems aredescribed by FIG. 6A, in which R₁₀₀, R₁₀₁, R₁₀₂, R₁₀₃, R₁₀₄ and R₁₀₅ areeach independently H, F, Cl, Br, I, OH, CN, NO₂ or a moiety thatincludes up to 16 carbon atoms and, optionally, one or more N, O, S or Fatoms; and X is S (see FIG. 6B) or O (see FIG. 6C).

In other embodiments, the compounds that include a 5-memberedheterocyclic ring system fused to one or more other ring systems aredescribed by FIG. 6D, in which R₁₀₆, R₁₀₇, R₁₀₈, R₁₀₉, R₁₁₀, R₁₁₁, R₁₁₂,R₁₁₃, R₁₁₄ and R₁₁₅ are each independently H, F, Cl, Br, I, OH, CN, NO₂or a moiety that includes up to 16 carbon atoms and, optionally, one ormore N, O, S or F atoms; and X is S (see FIG. 6E) or O (see FIG. 6F).

In certain embodiments, the compounds that include a 5-memberedheterocyclic ring system fused to one or more other ring systems aredescribed by FIG. 6G, in which R₁₁₈, R₁₁₉, R₁₂₀, R₁₂₁, R₁₂₃ are eachindependently H, F, Cl, Br, I, OH, CN, NO₂ or a moiety that includes upto 16 carbon atoms and, optionally, one or more N, O, S or F atoms; andR₁₂₃ is H or a moiety that includes up to 16 carbon atoms and,optionally, one or more N, O, S or F atoms.

In certain embodiments, the compounds that include a 5-memberedheterocyclic ring system fused to one or more other ring systems aredescribed by FIG. 6H, in which R₁₂₄, R₁₂₅, R₁₂₆ and R₁₂₇ are eachindependently H, F, Cl, Br, I, OH, CN, NO₂ or a moiety that includes upto 16 carbon atoms and, optionally, one or more N, O, S or F atoms; andR₁₂₈ and R₁₂₉ together define one or more rings that each include up to16 carbon atoms and, optionally, one or more N, O, S or F atoms. Forexample, the compounds of FIG. 6H can be described by FIG. 6I, in whichR₁₃₀, R₁₃₁, R₁₃₂ and R_(128′) are each independently H, F, Cl, Br, I,OH, CN, NO₂ or a moiety that includes up to 16 carbon atoms and,optionally, one or more N, O, S or F atoms; and R_(129′) is H, or amoiety that includes up to 16 carbon atoms and, optionally, one or moreN, O, S or F atoms.

In other certain embodiments, the compounds that include a 5-memberedheterocyclic ring system fused to one or more other ring systems aredescribed by FIG. 6J, in which R₁₃₄, R₁₃₅, R₁₃₆, R₁₃₇, R₁₄₀ and R₁₄₁ areeach independently H, F, Cl, Br, I, OH, CN, NO₂ or a moiety thatincludes up to 16 carbon atoms and, optionally, one or more N, O, S or Fatoms; and R₁₃₉ is H or a moiety that includes up to 16 carbon atomsand, optionally, one or more N, O, S or F atoms. For example and byreference to FIG. 6K, in specific embodiments, R₁₄₀ and R₁₄₁ togetherdefine a one or more rings that each include up to 16 carbon atoms and,optionally, one or more N, O, S or F atoms.

In some embodiments, the compounds that include a 5-memberedheterocyclic ring system fused to one or more other ring systems aredescribed by FIG. 6L, in which R₁₄₃, R₁₄₄, R₁₄₅, R₁₄₆ and R₁₄₇ are eachindependently H, F, Cl, Br, I, OH, CN, NO₂ or a moiety that includes upto 16 carbon atoms and, optionally, one or more N, O, S or F atoms; andX is O (see FIG. 6M) or S (see FIG. 6N).

In still other embodiments, the compounds that include a 5-memberedheterocyclic ring system fused to one or more other ring systems aredescribed by FIG. 6O, in which R₁₄₉, R₁₅₀, R₁₅₁ and R₁₅₂ are eachindependently H, F, Cl, Br, I, OH, CN, NO₂ or a moiety that includes upto 16 carbon atoms and, optionally, one or more N, O, S or F atoms; andR₁₅₃ is H or a moiety that includes up to 16 carbon atoms and,optionally, one or more N, O, S or F atoms.

In some embodiments, the compounds that can be utilized in any methoddescribed herein are pyridines or pyrimidines. For example, suchcompounds can be generally represented by those structures shown inFIGS. 7A-7D, and specifically exemplified in those structures shown inFIGS. 7E and 7F. Any described compound that is a pyridine or apyrimidine can be in neutral or salt form, e.g., a hydrochloride saltthereof.

In some embodiments, the pyridine compounds are described by FIG. 7A, inwhich R₁₅₅, R₁₅₆, R₁₅₇, R₁₅₉ and R₁₆₀ are each independently H, F, Cl,Br, I, OH, CN, NO₂ or a moiety that includes up to 16 carbon atoms and,optionally, one or more N, O, S or F atoms. In some specificembodiments, R₁₅₆ and R₁₅₇ (see FIG. 7B), or R₁₅₆ and R₁₅₇ and R₁₅₉ andR₁₆₀ (see FIG. 7C) together define a one or more rings that each includeup to 16 carbon atoms and, optionally, one or more N, O, S or F atoms.

In some embodiments, the pyrimidine compounds are described by FIG. 7D,in which R₁₆₁, R₁₆₂, R₁₆₃ and R₁₆₄ are each independently H, F, Cl, Br,I, OH, CN, NO₂ or a moiety that includes up to 16 carbon atoms and,optionally, one or more N, O, S or F atoms.

In some embodiments, the compounds that can be utilized in any methoddescribed herein are anilines or aniline derivatives. For example, suchcompounds can be generally represented by those structures shown inFIGS. 8A and 8B, and specifically exemplified in those structures shownin FIG. 8C. Any described compound that is a pyridine or a pyrimidinecan be in neutral or salt form.

In some embodiments, the aniline compounds are described by FIG. 8A, inwhich R₁₇₀, R₁₇₁, R₁₇₂, R₁₇₃ and R₁₇₄ are each independently H, F, Cl,Br, I, OH, CN, NO₂ or a moiety that includes up to 16 carbon atoms and,optionally, one or more N, O, S or F atoms; and R₁₇₅ and R₁₇₆ are eachindependently H or a moiety that includes up to 16 carbon atoms and,optionally, one or more N, O, S or F atoms.

In some embodiments, the aniline derivative compounds are described byFIG. 8B, in which R₁₇₈, R₁₇₉, R₁₈₀ and R₁₈₁ are each independently H, F,Cl, Br, I, OH, CN, NO₂, or a moiety that includes up to 16 carbon atomsand, optionally, one or more N, O, S or F atoms; and R₁₇₇ is H or amoiety that includes up to 16 carbon atoms and, optionally, one or moreN, O, S or F atoms.

In some embodiments, the compounds can have the formula delineated inFIG. 6G:

In some embodiments:

each of R₁₁₈, R₁₁₉, R₁₂₀, and R₁₂₁ is, independently selected from H,halo, OH, CN, NO₂, C₁-C₃ alkyl, C₁-C₃ haloalkyl, C₁-C₃ alkoxy, and C₁-C₃haloalkoxy;

R₁₂₂ is hydrogen or —Z—R^(a); wherein:

Z is O or a bond; and

R^(a) is:

-   -   (i) C₁-C₆ alkyl or C₁-C₆ haloalkyl, each of which is optionally        substituted with from 1-3 R^(b); or    -   (ii) C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, each of which is        optionally substituted with from 1-5 R^(c); or    -   (iii) C₇-C₁₁ aralkyl, or heteroaralkyl including 6-11 atoms,        each of which is optionally substituted with from 1-5 R^(c);    -   (iv) C₆-C₁₀ aryl or heteroaryl including 5-10 atoms, each of        which is optionally substituted with from 1-5 R^(d);

R₁₂₃ is:

-   -   (i) hydrogen; or    -   (ii) C₁-C₆ alkyl or C₁-C₆ haloalkyl, each of which is optionally        substituted with from 1-3 R^(b); or    -   (iii) C₆-C₁₀ aryl or heteroaryl including 5-10 atoms, each of        which is optionally substituted with from 1-5 R^(d); or    -   (iv) C₇-C₁₁ aralkyl, or heteroaralkyl including 6-11 atoms, each        of which is optionally substituted with from 1-5 R^(c); or    -   (v) —(C₁-C₆ alkyl)-Z¹—(C₆-C₁₀ aryl), wherein Z¹ is O, S, NH, or        N(CH₃); the alkyl portion is optionally substituted with from        1-3 R^(b); and the aryl portion is optionally substituted with        from 1-5 R^(d); or    -   (vi) —(C₁-C₆ alkyl)-Z²-(heteroaryl including 5-10 atoms),        wherein Z² is O, S, NH, or N(CH₃); the alkyl portion is        optionally substituted with from 1-3 R^(b); and the heteroaryl        portion is optionally substituted with from 1-5 R^(d); or    -   (vii) —(C₁-C₆ alkyl)-Z³—(C₃-C₁₀ cycloalkyl), wherein Z³ is O, S,        NH, or N(CH₃); the alkyl portion is optionally substituted with        from 1-3 R^(b); and the cycloalkyl portion is optionally        substituted with from 1-5 R^(c);

R^(b) at each occurrence is, independently:

-   -   (i) NH₂; NH(C₁-C₃ alkyl); N(C₁-C₃ alkyl)₂; hydroxy; C₁-C₆ alkoxy        or C₁-C₆ haloalkoxy; or    -   (ii) C₃-C₇ cycloalkyl optionally substituted with from 1-3        substituents independently selected from C₁-C₆ alkyl, NH₂;        NH(C₁-C₃ alkyl); N(C₁-C₃ alkyl)₂; hydroxy; C₁-C₆ alkoxy or C₁-C₆        haloalkoxy;

R^(c) at each occurrence is, independently:

-   -   (i) halo; NH₂; NH(C₁-C₃ alkyl); N(C₁-C₃ alkyl)₂; hydroxy; C₁-C₆        alkoxy; C₁-C₆ haloalkoxy; or oxo; or    -   (ii) C₁-C₆ alkyl or C₁-C₆ haloalkyl; and

R^(d) at each occurrence is, independently:

-   -   (i) halo; NH₂; NH(C₁-C₃ alkyl); N(C₁-C₃ alkyl)₂; hydroxy; C₁-C₆        alkoxy or C₁-C₆ haloalkoxy; nitro; —NHC(O)(C₁-C₃ alkyl); or        cyano; or    -   (ii) C₁-C₆ alkyl or C₁-C₆ haloalkyl.

Embodiments can include one or more of the following features.

Variables R₁₁₈, R₁₁₉, R₁₂₀, and R₁₂₁

In certain embodiments, each of R₁₁₈, R₁₁₉, R₁₂₀, and R₁₂₁ is hydrogen.In other embodiments, each of R₁₁₈, R₁₁₉, R₁₂₀, and R₁₂₁ isindependently selected from H, halo and NO₂. In still other embodiments,one of R₁₁₈, R₁₁₉, R₁₂₀, and R₁₂₁ (e.g., R₁₂₀) is halo, OH, CN, NO₂,C₁-C₃ alkyl, C₁-C₃ haloalkyl, C₁-C₃ alkoxy, or C₁-C₃ haloalkoxy (e.g.,halo, e.g., chloro; or NO₂); and the others are hydrogen (e.g., one ofR₁₁₈, R₁₁₉, R₁₂₀, and R₁₂₁ (e.g., R₁₂₀) is halo and NO₂, and the othersare hydrogen).

Variable R₁₂₂

In certain embodiments, R₁₂₂ can be —Z—R^(a). Embodiments can includeone or more of the following features.

Z can be O.

Z can be a bond.

R^(a) can be:

-   -   (i) C₁-C₆ alkyl or C₁-C₆ haloalkyl, each of which is optionally        substituted with from 1-3 R^(b); or    -   (iii) C₇-C₁₁ aralkyl, or heteroaralkyl including 6-11 atoms,        each of which is optionally substituted with from 1-5 R^(c).

For example, R^(a) can be:

-   -   (i) C₁-C₆ alkyl, each of which is optionally substituted with        from 1-3 R^(b);    -   or    -   (iii) C₇-C₁₁ aralkyl, which is optionally substituted with from        1-5 R^(c).

R^(a) can be C₇-C₁₁ aralkyl, or heteroaralkyl including 6-11 atoms, eachof which is optionally substituted with from 1-5 R^(c) (e.g., C₇-C₁₁aralkyl, which is optionally substituted with from 1-5 R^(c)). Forexample, R^(a) can be benzyl or phenethyl, in which the phenyl portionis optionally substituted with from 1-5 (e.g., 1-4, 1-3, 1-2, or 1,e.g., 1-2 or 1) R^(c) (e.g., halo (e.g., chloro); C₁-C₆ alkoxy (e.g.,OCH₃); or C₁-C₆ alkyl (e.g., CH₃). In certain embodiments, Z can be O.

R^(a) can be C₁-C₆ alkyl or C₁-C₆ haloalkyl, each of which is optionallysubstituted with from 1-3 R^(b) (e.g., C₁-C₆ alkyl, each of which isoptionally substituted with from 1-3 R^(b)). For example, R^(a) can beCH₃. In certain embodiments, Z can be a bond.

In certain embodiments, R₁₂₂ can be hydrogen.

Variable R₁₂₃

In certain embodiments, R₁₂₃ can be:

-   -   (iii) C₆-C₁₀ aryl or heteroaryl including 5-10 atoms, each of        which is optionally substituted with from 1-5 R^(d); or    -   (iv) C₇-C₁₁ aralkyl, or heteroaralkyl including 6-11 atoms, each        of which is optionally substituted with from 1-5 R^(c); or    -   (v) —(C₁-C₆ alkyl)-Z¹—(C₆-C₁₀ aryl), wherein Z¹ is O, S, NH, or        N(CH₃); the alkyl portion is optionally substituted with from        1-3 R^(b); and the aryl portion is optionally substituted with        from 1-5 R^(d).

For example, R₁₂₃ can be:

-   -   (iii) C₆-C₁₀ aryl, which is optionally substituted with from 1-5        R^(d); or    -   (iv) C₇-C₁₁ aralkyl, which is optionally substituted with from        1-5 R^(c); or    -   (v) —(C₁-C₆ alkyl)-Z¹—(C₆-C₁₀ aryl), wherein Z¹ is O, S, NH, or        N(CH₃); the alkyl portion is optionally substituted with from        1-3 R^(a); and the aryl portion is optionally substituted with        from 1-5 R^(d).

In embodiments, R₁₂₃ can be C₆-C₁₀ aryl or heteroaryl including 5-10atoms, each of which is optionally substituted with from 1-5 R^(d)(e.g., C₆-C₁₀ aryl, which is optionally substituted with from 1-5R^(d)). For example, R₁₂₃ can be phenyl, is optionally substituted withfrom 1-5 (e.g., 1-4, 1-3, 1-2, or 1) R^(d) (e.g., C₁-C₆ alkoxy, e.g.,OCH₃).

In embodiments, R₁₂₃ can be C₇-C₁₁ aralkyl, or heteroaralkyl including6-11 atoms, each of which is optionally substituted with from 1-5 R^(c)(e.g., C₇-C₁₁ aralkyl, which is optionally substituted with from 1-5R^(c)). For example, R₁₂₃ can be benzyl or phenethyl, in which thephenyl portion is optionally substituted with from 1-5 (e.g., 1-4, 1-3,1-2, or 1) R^(c) (e.g., halo (e.g., chloro); C₁-C₆ alkoxy (e.g., OCH₃);C₁-C₆ alkyl (e.g., CH₃); NH₂; or hydroxyl).

In embodiments, R₁₂₃ can be —(C₁-C₆ alkyl)-Z¹—(C₆-C₁₀ aryl), wherein Z¹is O, S, NH, or N(CH₃); the alkyl portion is optionally substituted withfrom 1-3 R^(b); and the aryl portion is optionally substituted with from1-5 R^(d). For example, R₁₂₃ can be —(CH₂)—Z¹-(phenyl), in which thephenyl portion is optionally substituted with from 1-5 (e.g., 1-4, 1-3,1-2, or 1) R^(d) (e.g., halo (e.g., chloro); C₁-C₆ alkoxy (e.g., OCH₃);C₁-C₆ alkyl (e.g., CH₃); NH₂; or hydroxyl).

A subset of compounds includes those in which:

R₁₂₂ is —Z—R^(a), in which R^(a) can be C₇-C₁₁ aralkyl, or heteroaralkylincluding 6-11 atoms, each of which is optionally substituted with from1-5 R^(c) (e.g., C₇-C₁₁ aralkyl, e.g., benzyl or phenethyl, which isoptionally substituted with from 1-5 R^(c)); and

R₁₂₃ can be C₆-C₁₀ aryl or heteroaryl including 5-10 atoms, each ofwhich is optionally substituted with from 1-5 R^(d) (e.g., C₆-C₁₀ aryl,e.g., phenyl, which is optionally substituted with from 1-5 R^(d)).

Embodiments can include one or more of the following features:

R^(c) and R^(d) can be as defined anywhere herein.

Z can be O.

Each of R₁₁₈, R₁₁₉, R₁₂₀, and R₁₂₁ is hydrogen. In other embodiments,each of R₁₁₈, R₁₁₉, R₁₂₀, and R₁₂₁ is independently selected from H,halo and NO₂. In still other embodiments, one of R₁₁₈, R₁₁₉, R₁₂₀, andR₁₂₁ (e.g., R₁₂₀) is halo, OH, CN, NO₂, C₁-C₃ alkyl, C₁-C₃ haloalkyl,C₁-C₃ alkoxy, or C₁-C₃ haloalkoxy (e.g., halo, e.g., chloro; or NO₂);and the others are hydrogen (e.g., one of R₁₁₈, R₁₁₉, R₁₂₀, and R₁₂₁(e.g., R₁₂₀) is halo and NO₂, and the others are hydrogen); e.g., one ofis halo (e.g., chloro) or nitro, e.g., halo (e.g., chloro), and theothers are hydrogen.

For example:

R₁₂₂ is —Z—R^(a), wherein Z is O, and R^(a) is C₇-C₁₁ aralkyl, which isoptionally substituted with from 1-5 R^(c); and

R₁₂₃ is C₆-C₁₀ aryl, which is optionally substituted with from 1-5R^(d); and

each of R₁₁₈, R₁₁₉, R₁₂₀, and R₁₂₁ can be hydrogen; or each of R₁₁₈,R₁₁₉, R₁₂₀, and R₁₂₁ can be, independently halo (e.g., chloro) or nitro,e.g., halo (e.g., chloro); or one of R₁₁₈, R₁₁₉, R₁₂₀, and R₁₂₁ can behalo (e.g., chloro) or NO₂, e.g., halo (e.g., chloro); and the othersare hydrogen.

As another example, Z is a bond, and the definitions in the aboveexample apply.

Examples of compounds having the formula delineated in FIG. 6G include:CP-0000489, CP-0000540, CP-0000550, CP-0000553, CP-0000554, CP-0000557,CP-0000571, CP-0047659, CP-0064483, CP-0066829, CP-0069961, CP-0074806,CP-0080773, CP-0091818, CP-0109953, CP-0105772, and CP-0193184.

Other examples of compounds having the formula delineated in FIG. 6Ginclude:

In some embodiments, the compounds can have the formula delineated inFIG. 6L:

In some embodiments:

X is O or S;

each of R₁₄₃, R₁₄₄, R₁₄₅, and R₁₄₆ is, independently selected from H,halo, OH, CN, NO₂, C₁-C₃ alkyl, C₁-C₃ haloalkyl, C₁-C₃ alkoxy, C₁-C₃haloalkoxy; and —NHC(O)(C₁-C₃ alkyl);

R₁₄₇ is NR^(e)R^(f), wherein one of R^(e) and R^(f) is hydrogen or C₁-C₃alkyl; and the other of R^(e) and R^(f) is:

-   -   (i) —C(O)R^(g); wherein R^(g) is C₆-C₁₀ aryl or heteroaryl        including 5-10 atoms, each of which is optionally substituted        with from 1-5 R^(h); or    -   (ii) C₁-C₃ alkyl;

or

R₁₄₇ is C₆-C₁₀ aryl or heteroaryl including 5-10 atoms, each of which isoptionally substituted with from 1-5 R^(h);

or

R₁₄₇ is —SCH₂R^(i), wherein R^(i) is:

-   -   (i) C₆-C₁₀ aryl or heteroaryl including 5-10 atoms, each of        which is optionally substituted with from 1-5 R^(h); or    -   (ii) —C(O)NR^(e)R^(f), wherein one of R^(e) and R^(f) is        hydrogen or C₁-C₃ alkyl; and the other of R^(e) and R^(f) is        —C(O)R^(g); wherein R^(g) is C₆-C₁₀ aryl or heteroaryl including        5-10 atoms, each of which is optionally substituted with from        1-5 R^(h); and

R^(h) at each occurrence is, independently:

-   -   (i) halo; NH₂; NH(C₁-C₃ alkyl); N(C₁-C₃ alkyl)₂; hydroxy; C₁-C₆        alkoxy or C₁-C₆ haloalkoxy; nitro; or cyano; or    -   (ii) C₁-C₆ alkyl or C₁-C₆ haloalkyl.

Embodiments can include one or more of the following features.

Variable X

X can be S.

X can be O.

Variables R₁₄₃, R₁₄₄, R₁₄₅, and R₁₄₆

In certain embodiments, each of R₁₄₃, R₁₄₄, R₁₄₅, and R₁₄₆ is hydrogen.In other embodiments, one of R₁₄₃, R₁₄₄, R₁₄₅, and R₁₄₆ is halo, OH, CN,NO₂, C₁-C₃ alkyl, C₁-C₃ haloalkyl, C₁-C₃ alkoxy, C₁-C₃ haloalkoxy; or—NHC(O)(C₁-C₃ alkyl); and the others are hydrogen.

Variable R₁₄₇

In certain embodiments, R₁₄₇ can be NR^(e)R^(f), wherein one of R^(e)and R^(f) is hydrogen or C₁-C₃ alkyl (e.g., hydrogen); and the other ofR^(e) and R^(f) is:

-   -   (i) —C(O)R^(g); wherein R^(g) is C₆-C₁₀ aryl or heteroaryl        including 5-10 atoms, each of which is optionally substituted        with from 1-5 R^(h); or    -   (ii) C₁-C₃ alkyl.

In embodiments, R₁₄₇ can be NR^(e)R^(f), wherein one of R^(e) and R^(f)is hydrogen or C₁-C₃ alkyl (e.g., hydrogen); and the other of R^(e) andR^(f) is —C(O)R^(g); wherein R^(g) is C₆-C₁₀ aryl or heteroarylincluding 5-10 atoms, each of which is optionally substituted with from1-5 R^(h).

By way of example, R^(g) can be phenyl, which is optionally substitutedwith from 1-5 (e.g., 1-4, 1-3, 1-2, or 1) R^(h) (e.g., halo (e.g.,chloro); C₁-C₆ alkoxy (e.g., OCH₃); or C₁-C₆ alkyl (e.g., CH₃)).

As another example, R^(g) can be heteroaryl including 5-6 (e.g., 5)atoms, which is optionally substituted with from 1-2 (e.g., 1) R^(h)(e.g., C₁-C₆ alkyl (e.g., CH₃)).

In certain embodiments:

X can be S; and

R₁₄₇ can be NR^(e)R^(f), wherein one of R^(e) and R^(f) is hydrogen orC₁-C₃ alkyl (e.g., hydrogen); and the other of R^(e) and R^(f) is:

-   -   (i) —C(O)R^(g); wherein R^(g) is C₆-C₁₀ aryl or heteroaryl        including 5-10 atoms, each of which is optionally substituted        with from 1-5 R^(h); or    -   (ii) C₁-C₃ alkyl;

(e.g., one of R^(e) and R^(f) is hydrogen or C₁-C₃ alkyl (e.g.,hydrogen); and the other of R^(e) and R^(f) is —C(O)R^(g); wherein R^(g)is C₆-C₁₀ aryl or heteroaryl including 5-10 atoms, each of which isoptionally substituted with from 1-5 R^(h)).

R^(g) and R^(h) can be as defined anywhere herein.

In certain embodiments, each of R₁₄₃, R₁₄₄, R₁₄₅, and R₁₄₆ is hydrogen.In other embodiments, one of R₁₄₃, R₁₄₄, R₁₄₅, and R₁₄₆ is halo, OH, CN,NO₂, C₁-C₃ alkyl, C₁-C₃ haloalkyl, C₁-C₃ alkoxy, C₁-C₃ haloalkoxy; or—NHC(O)(C₁-C₃ alkyl); and the others are hydrogen.

Examples of compounds having the formula delineated in FIG. 6L include:CP-0064917, CP-0067233, CP-0068578, CP-0103014, CP-0105777, CP-0107060,CP-0029300, CP-0079983, and CP-0103978.

In some embodiments, the compounds can have the formula delineated inFIG. 6A:

In some embodiments:

X is O or S;

each of R₁₀₀, R₁₀₁, R₁₀₂, and R₁₀₃, is, independently selected from H,halo, OH, CN, NO₂, C₁-C₃ alkyl, C₁-C₃ haloalkyl, C₁-C₃ alkoxy, C₁-C₃haloalkoxy; or

any two adjacent pairs of R₁₀₀, R₁₀₁, R₁₀₂, and R₁₀₃, together with thecarbon atoms to which they are attached form a fused heterocyclic ringincluding 5 or 6 total ring atoms; wherein the heterocyclic ring isoptionally substituted with from 1-3 substituents independently selectedfrom C₁-C₃ alkyl and oxo;

R₁₀₄ is —C(O)NR^(j)R^(k), wherein one of and R^(k) is hydrogen or C₁-C₃alkyl; and the other of and R^(k) is:

-   -   (i) C₁-C₈ alkyl, optionally substituted with a 5-6 heterocyclyl;        or    -   (ii) heteroaryl including 5-6 atoms, which is optionally        substituted with from 1-5 substituents independently selected        from halo, OH, CN, NO₂, C₁-C₃ alkyl, C₁-C₃ haloalkyl, C₁-C₃        alkoxy, C₁-C₃ haloalkoxy; —C(O)NH₂; —NHC(O)(C₁-C₃ alkyl); and a        fused C₅-C₆ cycloalkyl ring;

or

R¹⁰⁴ is heteroaryl including 5-6 atoms, which is optionally substitutedwith from 1-5 substituents independently selected from halo, OH, CN,NO₂, C₁-C₃ alkyl, C₁-C₃ haloalkyl, C₁-C₃ alkoxy, C₁-C₃ haloalkoxy;—C(O)NH₂; —NHC(O)(C₁-C₃ alkyl); and

R₁₀₅ is halo or C₁-C₃ alkyl.

Embodiments can include one or more of the following features.

Variable X

X can be S.

X can be O.

Variables R₁₀₀, R₁₀₁, R₁₀₂, and R₁₀₃

In certain embodiments, each of R₁₀₀, R₁₀₁, R₁₀₂, and R₁₀₃ is hydrogen.In other embodiments, one of R₁₀₀, R₁₀₁, R₁₀₂, and R₁₀₃ is halo, OH, CN,NO₂, C₁-C₃ alkyl, C₁-C₃ haloalkyl, C₁-C₃ alkoxy, C₁-C₃ haloalkoxy; or—NHC(O)(C₁-C₃ alkyl); and the others are hydrogen.

Variable R₁₀₄

In certain embodiments, R₁₀₄ is —C(O)NR^(j)R^(k), wherein one of R^(j)and R^(k) is hydrogen or C₁-C₃ alkyl (e.g., hydrogen); and the other ofR^(j) and R^(k) is:

-   -   (i) C₁-C₈ alkyl, optionally substituted with a 5-6 heterocyclyl;        or    -   (ii) heteroaryl including 5-6 atoms, which is optionally        substituted with from 1-5 substituents independently selected        from halo, OH, CN, NO₂, C₁-C₃ alkyl, C₁-C₃ haloalkyl, C₁-C₃        alkoxy, C₁-C₃ haloalkoxy; cyano; —C(O)NH₂; —NHC(O)(C₁-C₃ alkyl);        C₁-C₃ alkyl; C₁-C₃ alkyl; and a fused C₅-C₆ cycloalkyl ring.

By way of example, one of R^(j) and R^(k) is hydrogen or C₁-C₃ alkyl(e.g., hydrogen); and the other of R^(j) and R^(k) is heteroarylincluding 5-6 atoms, which is optionally substituted with from 1-5substituents independently selected from halo, OH, CN, NO₂, C₁-C₃ alkyl,C₁-C₃ haloalkyl, C₁-C₃ alkoxy, C₁-C₃ haloalkoxy; —C(O)NH₂; —NHC(O)(C₁-C₃alkyl); and a fused C₅-C₆ cycloalkyl ring.

Variable R₁₀₅

R₁₀₅ can be chloro or CH₃.

Examples of compounds having the formula delineated in FIG. 6A include:CP-0079175, CP-0087336, CP-0064314, CP-0068577, and CP-0102404.

In some embodiments, the compounds can have the formula delineated inFIG. 3A:

In some embodiments:

R₄₃ is C₆-C₁₀ aryl or heteroaryl including 5-10 atoms, each of which isoptionally substituted with from 1-5 R^(m);

R₄₄ is:

-   -   (i) C₆-C₁₀ aryl or heteroaryl including 5-10 atoms, each of        which is optionally substituted with from 1-5 R^(m); or    -   (ii) —Z⁴—(C₁-C₆ alkyl), wherein:    -   Z⁴ is a bond or NH; and    -   the C₁-C₆ alkyl is substituted with one of the following:        -   (a) heterocyclyl including 5-6 atoms, which is optionally            substituted with from 1-3 substituents independently            selected from oxo and C₁-C₆ alkyl; or        -   (b) phenoxy, which is optionally substituted with from 1-5            R^(m); and

R^(m) at each occurrence is, independently:

-   -   (i) halo; NH₂; NH(C₁-C₃ alkyl); N(C₁-C₃ alkyl)₂; hydroxy; C₁-C₆        alkoxy or C₁-C₆ haloalkoxy; nitro; or cyano; or    -   (ii) C₁-C₆ alkyl or C₁-C₆ haloalkyl.

Embodiments can include one or more of the following features.

Variable R₄₃

In certain embodiments, R₄₃ can be C₆-C₁₀ aryl, which is optionallysubstituted with from 1-5 R^(m). For example, R₄₃ can be phenyl, whichis optionally substituted with from 1-5 (e.g., 1-4, 1-3, 1-2, or 1)R^(m) (e.g., C₁-C₆ alkyl (e.g., CH₃)).

In certain embodiments, R₄₃ can be heteroaryl including 5-6 atoms, eachof which is optionally substituted with from 1-5 R^(m).

Variable R₄₄

In certain embodiments, R₄₄ can be C₆-C₁₀ aryl, which is optionallysubstituted with from 1-5 R^(m). For example, R₄₄ can be phenyl, whichis optionally substituted with from 1-5 (e.g., 1-4, 1-3, 1-2, or 1)R^(m) (e.g., halo (e.g., chloro); C₁-C₆ alkoxy (e.g., OCH₃); or C₁-C₆alkyl (e.g., CH₃)).

Examples of compounds having the formula delineated in FIG. 3A include:CP-0067108, CP-0067246, CP-0068395, CP-0068929, CP-0068961, CP-0070164,CP-0070367, CP-0079642, CP-0104904, and CP-0130665.

In some embodiments, the compounds can have the formula delineated inFIG. 3U:

In some embodiments:

Each of R₆₇ and R₆₈ is, independently:

-   -   (i) hydrogen; or    -   (ii) C₆-C₁₀ aryl or heteroaryl including 5-10 atoms, each of        which is optionally substituted with from 1-5 R^(n); or    -   (iii) NH₂; or    -   (iv) —C(O)(C₁-C₆ alkyl);

R₆₉ is NR^(o)R^(p), wherein one of R^(o) and R^(p) is hydrogen or C₁-C₃alkyl; and the other of R^(o) and R^(p) is:

-   -   (i) hydrogen; or    -   (ii) C₆-C₁₀ aryl or heteroaryl including 5-6 atoms, which is        optionally substituted with from 1-5 R^(a); or    -   (iii) —C(O)(C₁-C₆ alkyl), wherein the C₁-C₆ alkyl is substituted        with phenoxy that is optionally substituted with from 1-5 R^(n);

R^(n) at each occurrence is, independently:

-   -   (i) halo; NH₂; NH(C₁-C₃ alkyl); N(C₁-C₃ alkyl)₂; hydroxy; C₁-C₆        alkoxy or C₁-C₆ haloalkoxy; nitro; or cyano; or    -   (ii) C₁-C₆ alkyl or C₁-C₆ haloalkyl; or    -   (iii) phenyl.

Embodiments can include one or more of the following features.

Variables R₆₇ and R₆₈

In certain embodiments, one of R₆₇ and R₆₈ is C₆-C₁₀ aryl or heteroarylincluding 5-10 atoms, each of which is optionally substituted with from1-5 R^(n); and the other is hydrogen.

Variable R₆₉

In certain embodiments, one of R^(o) and R^(p) is hydrogen or C₁-C₃alkyl (e.g., hydrogen); and the other of R^(o) and R^(p) is C₆-C₁₀ arylor heteroaryl including 5-6 atoms, which is optionally substituted withfrom 1-5

Examples of compounds having the formula delineated in FIG. 3U include:CP-0063182, CP-0071862, CP-0072036, CP-0105343, CP-0122949, andCP-0134381.

In some embodiments, the compounds can have the formula delineated inFIG. 3E:

In some embodiments:

X is O or S;

R₅₀ and R₅₃ are each, independently:

-   -   (i) hydrogen; or    -   (ii) —C(O)R^(q); or    -   (iii) C₆-C₁₀ aryl or heteroaryl including 5-10 atoms, each of        which is optionally substituted with from 1-5 R^(r);    -   provided that at least one of R₅₀ and R₅₃ is other than        hydrogen;

R₅₁ and R₅₂ are each, independently, hydrogen or halo;

R^(q) is:

-   -   (i) C₁-C₆ alkyl; or    -   (ii) —NR^(s)R^(t); wherein:        -   (a) one of R^(s) and R^(t) is hydrogen, and the other is            C₆-C₁₀ aryl or heteroaryl including 5-10 atoms, each of            which is optionally substituted with from 1-5 R^(r); C₁-C₆            alkyl, which is substituted with phenoxy that is optionally            substituted with from 1-5 R^(r); or —O—N═C(NH₂)(C₆-C₁₀            aryl), wherein the aryl portion is optionally substituted            with from 1-5 R^(r); or        -   (b) R^(s) and R^(t), together with the nitrogen atom to            which each is attached forms a heterocyclyl including 5-6            atoms; or    -   (iii) —NH—C(O)(C₆-C₁₀ aryl), wherein the aryl portion is        optionally substituted with from 1-5 R^(r); and

R^(r) at each occurrence is, independently:

-   -   (i) halo; NH₂; NH(C₁-C₃ alkyl); N(C₁-C₃ alkyl)₂; hydroxy; C₁-C₆        alkoxy or C₁-C₆ haloalkoxy; nitro; or cyano; or    -   (ii) C₁-C₆ alkyl or C₁-C₆ haloalkyl.

In certain embodiments, one of R₅₀ and R₅₃ is —C(O)R^(q); and the otherof R₅₀ and R₅₃ is hydrogen or C₆-C₁₀ aryl or heteroaryl including 5-10atoms, each of which is optionally substituted with from 1-5 R^(r). Inembodiments, R^(q) can be —NR^(s)R^(t).

Examples of compounds having the formula delineated in FIG. 3E include:CP-0061777, CP-0066008, CP-0072253, CP-0099289, CP-0008545, CP-0060852,CP-0072156, CP-0072271, CP-0104766, and CP-0110352.

In some embodiments, the compounds can have the formula delineated inFIG. 3N:

In some embodiments:

each of R₆₁, R₆₂, and R₆₄ is, independently:

-   -   (i) hydrogen; or    -   (ii) C₆-C₁₀ aryl or heteroaryl including 5-10 atoms, each of        which is optionally substituted with from 1-5 R^(u); or    -   (iii) —NH—C(O)(C₆-C₁₀ aryl), wherein the aryl portion is        optionally substituted with from 1-5 R^(u); or    -   (iv) —C(O)NR^(v)R^(w), wherein one of R^(v) and R^(w) is        hydrogen; and the other of R^(v) and R^(w) is C₆-C₁₀ aryl, which        is optionally substituted with from 1-5 R^(u); or C₇-C₁₁        aralkyl, which is optionally substituted with oxo; or    -   (v) NH₂ or hydroxymethyl;

R₆₃ is:

-   -   (i) hydrogen; or    -   (ii) C₆-C₁₀ aryl or heteroaryl including 5-10 atoms, each of        which is optionally substituted with from 1-5 R^(u); or    -   (iii) C₁-C₆ alkyl; and

R^(u) at each occurrence is, independently:

-   -   (i) halo; NH₂; NH(C₁-C₃ alkyl); N(C₁-C₃ alkyl)₂; hydroxy; C₁-C₆        alkoxy or C₁-C₆ haloalkoxy; nitro; or cyano; or    -   (ii) C₁-C₆ alkyl or C₁-C₆ haloalkyl.

In certain embodiments, two of R₆₁, R₆₂, and R₆₄ are other thanhydrogen.

Examples of compounds having the formula delineated in FIG. 3N include:CP-0000477, CP-0063375, CP-0064231, CP-0065105, CP-0070844, CP-0070886,and CP-0104765.

In some embodiments, the compounds can have the formula delineated inFIG. 3V.

In certain embodiments, R₇₀ can be an amide (i.e., having the generalformula —C(O)NRR′) or reverse amide (i.e., having the general formula—NR″C(O)R′″) as described anywhere herein.

In certain embodiments, R₇₁ can be hydrogen.

In certain embodiments, R₇₂ can be:

-   -   (i) C₁-C₆ alkyl; or    -   (ii) C₆-C₁₀ aryl or heteroaryl including 5-10 atoms, each of        which is optionally substituted with from 1-5 substituents        independently selected from halo; NH₂; NH(C₁-C₃ alkyl); N(C₁-C₃        alkyl)₂; hydroxy; C₁-C₆ alkoxy or C₁-C₆ haloalkoxy; nitro;        cyano; C₁-C₆ alkyl; and C₁-C₆ haloalkyl.

Examples of compounds having the formula delineated in FIG. 3V include:CP-0065665, CP-0075627, and CP-0075656.

In some embodiments, the compounds can have the formula delineated inFIG. 7D.

In certain embodiments, the pyrimidine ring can be substituted with 1-2substituents independently selected from:

-   -   (i) heterocyclyl including 5-6 atoms; or    -   (ii) C₆-C₁₀ aryl or heteroaryl including 5-10 atoms, each of        which is optionally substituted with from 1-5 substituents        independently selected from halo; NH₂; NH(C₁-C₃ alkyl); N(C₁-C₃        alkyl)₂; hydroxy; C₁-C₆ alkoxy or C₁-C₆ haloalkoxy; nitro;        cyano; C₁-C₆ alkyl; and C₁-C₆ haloalkyl.

In other embodiments, the pyrimidine ring can be substituted with afused ring.

Examples of compounds having the formula delineated in FIG. 7D include:CP-0059547, CP-0059563, CP-0059642, CP-0064382, CP-0067053, CP-0072720,and CP-0079810.

In some embodiments, the compounds can have the formula delineated inFIG. 7A.

In certain embodiments, the pyridine ring can be substituted with anamide or reverse amide as described anywhere herein.

In other embodiments, the pyrimidine ring can be substituted with one ormore fused ring.

Examples of compounds having the formula delineated in FIG. 7A include:CP-0060729, CP-0066751, CP-0069934, CP-0076627, CP-0080276, CP-0089966,CP-0029278, and CP-0130586.

Mixtures of any of the compounds described herein can also be utilizedin any method described herein.

Methods of Synthesis

The compounds of the present invention can be obtained commercially fromsuppliers such as Bionet, Maybridge, Chemdiv, ChemBridge, Peakdale,IFLAB/Life Chemicals, Enamine, Microsource, or Timtec. Alternatively orin addition, the compounds described herein can be synthesized accordingto methods described herein (or variations thereof) and/or conventional,organic chemical synthesis methods from commercially available startingmaterials and reagents or from starting materials and reagents that canbe prepared according to conventional organic chemical synthesismethods. The compounds described herein can be separated from a reactionmixture and further purified by a method such as column chromatography,high-performance liquid chromatography (HPLC), or recrystallization. Ascan be appreciated by the skilled artisan, further methods ofsynthesizing the compounds of the formulae herein will be evident tothose skilled in the art. Additionally, the various synthetic steps maybe performed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in Larock, Comprehensive OrganicTransformations, 2d. ed., Wiley-VCH Publishers (1999); Wuts and Greene,Protective Groups in Organic Synthesis, 4th Ed., John Wiley and Sons(2007); Fieser and Fieser, Fieser and Fieser's Reagents for OrganicSynthesis, John Wiley and Sons (1994); and Paquette, ed., Encyclopediaof Reagents for Organic Synthesis, John Wiley and Sons (1995), andsubsequent editions thereof

Benzimidazole-Containing Compounds

Compounds having the formula delineated in FIG. 6G can be obtainedcommercially or synthesized using conventional synthetic methods. Forexample, compounds CP-0000489, CP-0000540, CP-0000550, CP-0000553,CP-0000554, CP-0000557, CP-0000571, CP-0047659, CP-0064483, CP-0066829,CP-0069961, CP-0074806, CP-0080773, CP-0091818, CP-0105772, andCP-0109953 were obtained commercially from the suppliers provided inTable 1 (Entries 1-17). Other benzimidazoles 1-14 (Scheme 1) describedin the present disclosure can be obtained commercially.

Other compounds having the formula delineated in FIG. 6G can beobtained, e.g., using the chemistries described in Kokare et al.,Protein & Peptide Letters, 14:259-263, 2007 which describes thesynthesis of CP-0000540. Benzimidazole analogs incorporating changes tothe specific portions of the molecule can be prepared according toScheme 2 utilizing well established chemistry. In cases where a1H-benzimidazole intermediate is commercially available, alkylationreactions can be carried out to introduce the R₁ substituent (Route A).Other elaborations of the commercial benzimidazoles can be utilized toinstall various substituents at different positions of the molecule.However, for unsymmetrically substituted 1H-benzimidazoles two otherroutes can be used. In cases of where R₃ is an electron-withdrawinggroup, nucleophilic aromatic substitution of 2-fluoronitrobenzenes canbe utilized (Route B) to give 2-aminonitrobenzene intermediates. Forother analogs, alkylation of 2-aminonitrobenzenes (Route C) to introducethe R₁ group can be pursued to give the same intermediate. The reductionof the nitro group to the amino group can be carried out withestablished reduction protocols. Oxidative cyclization of such1,2-diamines with aldehydes or condensations with carboxylic acids willgive the desired benzimidazole analogs.

Benzothiazole-Containing Compounds

Compounds having the formula delineated in FIG. 6L (X═S) can be obtainedcommercially or synthesized using conventional synthetic methods. Forexample, compounds CP-0064917, CP-0067233, CP-0068578, CP-0103014,CP-0105777, and CP-0107060 were obtained commercially from the suppliersprovided in Table 1 (Entries 18-23). Compounds having the formuladelineated in FIG. 6L (X═S) can be obtained, e.g., by the cyclization ofortho-halo benzamides using Lawesson's reagent or via the oxidation ofthioanilides. Other compounds having the formula delineated in FIG. 6L(X═S) can also be obtained, e.g., using the chemistries described inSong et al., Eur. J. Med. Chem. 43(7):1519-1524, 2008.

Benzoxazole-Containing Compounds

Compounds having the formula delineated in FIG. 6L (X═O) can be obtainedcommercially or synthesized using conventional synthetic methods. Forexample, compounds CP-0029300, CP-0079983, and CP-0103978 were obtainedcommercially from the suppliers provided in Table 1 (Entries 24-27).Other compounds having the formula delineated in FIG. 6L (X═O) can beobtained, e.g., using the chemistries described in Boyd, Sci. Synth.11:481-492, 2002.

Quinazolinone-Containing Compounds

Quinazolinone derivatives included in the FIG. 2G, can be obtainedcommercially or synthesized using conventional synthetic methods. Forexample, compounds CP-0034360 and CP-0036187 were obtained commerciallyfrom the suppliers provided in Table 1 (Entries 27-28). Other compoundshaving the formula delineated in FIG. 2C can be obtained, e.g., usingthe chemistries described in Connolly et al., Tetrahedron61(43):10153-10202, 2005.

Benzimidazopyrimidine-Containing Compounds

Benzimidazopyrimidine compounds having the formula delineated in FIG. 6Ican be obtained commercially or synthesized using conventional syntheticmethods. For example, compounds CP-0050095 and CP-0131763 were obtainedcommercially from the suppliers provided in Table 1 (Entries 29-30).

Benzofuran-Containing Compounds

Compounds having the formula delineated in FIG. 6A (X═O) can be obtainedcommercially or synthesized using conventional synthetic methods. Forexample, compounds CP-0079175 and CP-0087336 were obtained commerciallyfrom the suppliers provided in Table 1 (Entries 31-32). Other compoundshaving the formula delineated in FIG. 6A (X═O) can be obtained, e.g.,using the chemistries described in Hou, et al., Progress in HeterocyclicChemistry 17:142-171, 2005.

Benzothiophene-Containing Compounds Compounds having the formuladelineated in FIG. 6A (X═S) can be obtained commercially or synthesizedusing conventional synthetic methods. For example, compounds CP-0064314,CP-0068577, and CP-0102404 were obtained commercially from the suppliersprovided in Table 1 (Entries 33-35). Other compounds having the formuladelineated in FIG. 6A (X═S) can be obtained, e.g., using the chemistriesdescribed in either Bravo et al., J. Heterocyclic Chem., 7(4):967-8,1970, or Rayner et al., Sci. Synth. 10:155-181, 2005.

Indole-Containing Compounds

Compounds having the formula delineated in FIG. 6J can be obtainedcommercially or synthesized using conventional synthetic methods. Forexample, compounds CP-0010539, CP-0072096, CP-0078448, and CP-0103978were obtained commercially from the suppliers provided in Table 1(Entries 36-38). Other compounds having the formula delineated in FIG.6J can be obtained, e.g., using the chemistries described in Humphrey etal., Chem. Rev., 106(7):2875-2911, 2006.

Quinoline-Containing Compounds

Quinolines derivatives included in the FIG. 7E and 7F, can be obtainedcommercially or synthesized using conventional synthetic methods. Forexample, compounds CP-0072092 and CP-0087799 were obtained commerciallyfrom the suppliers provided in Table 1 (Entries 39-40). Other quinolinecompounds can be obtained, e.g., using the chemistries described inLarsen et al., Sci. Synth. 15:389-549, 2005.

Benzotriazole-Containing Compounds

Compounds having the formula delineated in FIG. 6O can be obtainedcommercially or synthesized using conventional synthetic methods. Forexample, compounds CP-0009883 and CP-0070871 were obtained commerciallyfrom the suppliers provided in Table 1 (Entries 41-42). Other compoundshaving the formula delineated in FIG. 6O can be obtained, e.g., usingthe chemistries described in Katritzky et al., Chem. Rev. 98(2):409-548,1998.

The coumarin-, benzopyran-, tetrahydroquinoline-, benzopyranone-, andbenzopyrazine-containing compounds of the invention can be obtainedcommercially or synthesized using conventional synthetic methods. Forexample, compounds CP-0063508, CP-0000928, CP-0005069, CP-0096433, andCP-0045061 included in the FIG. 1, were obtained from the suppliersprovided in Table 1 (Entries 43-47). Other coumarin-, benzopyran-,tetrahydroquinoline-, benzopyranone-, and benzopyrazine-containingcompounds can be obtained, e.g., using the chemistries described inBorges et al., Curr. Med. Chem. 12(8):887-916, 2005; Schweizer et al.,Chemistry of Heterocyclic Compounds 31:11-139, 1977; Katritzky et al.,Tetrahedron 52(48):15031-15070, 1996; Williams et al., Sci. Synth.14:347-638, 2003; Kress et al., Progress in Heterocyclic Chemistry4:186-203, 1992.

Pyridine-Containing Compounds

Compounds having the formula delineated in FIG. 7A can be obtainedcommercially or synthesized using conventional synthetic methods. Forexample, compounds CP-0060729, CP-0066751, CP-0069934, CP-0076627,CP-0080276, CP-0089966, CP-0029278, and CP-0130586 were obtainedcommercially from the suppliers provided in Table 1 (Entries 48-55).Other compounds having the formula delineated in FIG. 7A can beobtained, e.g., using the chemistries described in either Li et al.,Bioorg. & Med. Chem. Lett. 17(8):2347-2350, 2007, or in Spitzner et al.,Sci. Synth. 15:11-255, 2005.

Pyrimidine-Containing Compounds

Compounds having the formula delineated in FIG. 7D can be obtainedcommercially or synthesized using conventional synthetic methods. Forexample, compounds CP-0059547, CP-0059563, CP-0059642, CP-0064382,CP-0067053, CP-0072720, and CP-0079810 were obtained commercially fromthe suppliers provided in Table 1 (Entries 56-62). Other compoundshaving the formula delineated in FIG. 7D can be obtained, e.g., usingthe chemistries described in either Luo et al., Tetrahedron Lett.43(33), 5739-5742, 2002, or von Angerer et al., Sci. Synth. 16:379-572,2004.

Furan-Containing Compounds

Compounds having the formula delineated in FIG. 3E (X═O) can be obtainedcommercially or synthesized using conventional synthetic methods. Forexample, compounds CP-0061777, CP-0066008, CP-0072253, and CP-0099289were obtained commercially from the suppliers provided in Table 1(Entries 63-66). Other compounds having the formula delineated in FIG.3E (X═O) can be obtained, e.g., using the chemistries described ineither Kort et al., J. Med. Chem. 51(3):407-416, 2008, or Konig et al.,Sci. Synth. 9:183-286, 2001.

Thiophene-Containing Compounds

Compounds having the formula delineated in FIG. 3E (X═S) can be obtainedcommercially or synthesized using conventional synthetic methods. Forexample, compounds CP-0008545, CP-0060852, CP-0072156, CP-0072271,CP-0104766, and CP-0110352 were obtained commercially from the suppliersprovided in Table 1 (Entries 67-72). Other compounds having the formuladelineated in FIG. 3E (X═S) can be obtained, e.g., using the chemistriesdescribed in either Kaizerman et al., J. Med. Chem. 46(18):3914-3929,2003, or Schatz et al., Sci. Synth. 10:287-392, 2001.

Thiazole-Containing Compounds

Compounds having the formula delineated in FIG. 3U can be obtainedcommercially or synthesized using conventional synthetic methods. Forexample, compounds CP-0063182, CP-0071862, CP-0072036, CP-0105343,CP-0122949, and CP-0134381 were obtained commercially from the suppliersprovided in Table 1 (Entries 73-78). Other compounds having the formuladelineated in FIG. 3U can be obtained, e.g., using the chemistriesdescribed in either Narayana et al., Phosphorus, Sulfur and Silicon andthe Related Elements 181(6):1381-1389, 2006, or Kikelj et al., Sci.Synth. 11:627-806, 2002.

Pyrazole-Containing Compounds

Compounds having the formula delineated in FIG. 3N can be obtainedcommercially or synthesized using conventional synthetic methods. Forexample, compounds CP-0000477, CP-0063375, CP-0064231, CP-0065105,CP-0070844, CP-0070886, and CP-0104765 were obtained commercially fromthe suppliers provided in Table 1 (Entries 79-85). Other compoundshaving the formula delineated in FIG. 3N can be obtained, e.g., usingthe chemistries described in either McKeown et al., Bioorg. & Med. Chem.Lett., 16(18):4767-4771, 2006, or Stanovnik, et al., Sci. Synth.12:15-226, 2003.

Isoxazole-Containing Compounds

Compounds having the formula delineated in FIG. 3V can be obtainedcommercially or synthesized using conventional synthetic methods. Forexample, compounds CP-0065665, CP-0075627, and CP-0075656 were obtainedcommercially from the suppliers provided in Table 1 (Entries 86-88).Other compounds having the formula delineated in FIG. 3V can beobtained, e.g., using the chemistries described in Wakefield, Sci.Synth. 11:229-288, 2002.

Oxadiazole-Containing Compounds

Compounds having the formula delineated in FIG. 3A can be obtainedcommercially or synthesized using conventional synthetic methods. Forexample, compounds CP-0067108, CP-0067246, CP-0068395, CP-0068929,CP-0068961, CP-0070164, CP-0070367, CP-0079642, CP-0104904, andCP-0130665 were obtained commercially from the suppliers provided inTable 1 (Entries 89-98). Other compounds having the formula delineatedin FIG. 3A can be obtained, e.g., using the chemistries described ineither Grant et al., J. Org. Chem. 73(18):7219-7223, 2008, or Hemming,et al., Sci. Synth. 13:127-184, 2004.

Benzamide-Containing Compounds

Compounds having the formula delineated in FIG. 2A can be obtainedcommercially or synthesized using conventional synthetic methods. Forexample, compounds CP-0005186, CP-0007991, and CP-0061566 were obtainedcommercially from the suppliers provided in Table 1 (Entries 99-101).Other compounds having the formula delineated in FIG. 2A can be obtainedusing the methods known to one skilled in the art e.g., by acondensation of the corresponding benzoic acid and an amine.

The 1,3,4-oxadiazole-, triazoline-, pyrazoline-, dihydropyridone-,triazole-, indoline-, and imidazotriazine-containing compounds can beobtained commercially or synthesized using conventional syntheticmethods. For example, compounds CP-0062030, CP-0007994, CP-0039073,CP-0004116, CP-0061401, CP-0064286, CP-0110644, and CP-0051092 wereobtained commercially from the suppliers provided in Table 1 (Entries102-109).

TABLE 1 Supplier Com- Entry Number and (Supplier pound StructureName^(A) ID) CP- 0000489

1. 4-(4-chlorophenyl)-1- (5H-pyrimido[5,4- b]indol-4-yl)-1H-pyrazol-3-amine BIONET (bionet-7F- 307S) CP- 0000540

2. 6-chloro-1-(2- chlorobenzyloxy)-2- phenyl-1H- benzo[d]imidazoleBIONET (bionet-9F- 327S) CP- 0000550

3. 6-chloro-1-(2- chlorobenzyloxy)-2-(4- methoxyphenyl)-1H-benzo[d]imidazole BIONET (bionet-10F- 310S) CP- 0000553

4. 6-chloro-2-(4- methoxyphenyl)-1-(4- methylbenzyloxy)-1H-benzo[d]imidazole BIONET (bionet-10F- 324S) CP- 0000554

5. 6-chloro-1-(3,5- dimethylbenzyloxy)-2-(4- methoxyphenyl)-1H-benzo[d]imidazole BIONET (bionet-10F- 325S) CP- 0000557

6. 6-chloro-1-(4- methoxybenzyloxy)-2-(4- methoxyphenyl)-1H-benzo[d]imidazole BIONET (bionet-10F- 350S) CP- 0000571

7. 1-(4-methylbenzyloxy)- 6-nitro-2-phenyl-1H- benzo[d]imidazole BIONET(bionet-11F- 314S) CP- 0047659

8. 4-(1H-benzo[d] imidazol-2-yl)phenol CHEMDIV (4385-2057) CP- 0064483

9. 2,5-dichloro-N-((1- methyl-1H-benzo[d] imidazol-2- yl)methyl)anilineCHEMDIV (3546-0621) CP- 0066829

10. 4-(2-(1-methyl-1H- benzo[d]imidazol-2- yl)ethyl)aniline CHEMDIV(4432-2284) CP- 0069961

11. 2-((2-methoxyphenoxy) methyl)-1H- benzo[d]imidazole CHEMDIV (G856-0617) CP- 0074806

12. 2-((4- fluorophenoxy)methyl)- 1-methyl-1H- benzo[d]imidazole CHEMDIV(C147- 0180) CP- 0080773

13. 2-(phenylthiomethyl)- 1H-benzo[d]imidazole BIONET (bionet_8J- 311S)CP- 0091818

14. 3-(6-methyl-1H- benzo[d]imidazol-2-yl)- 2H-chromen-2-imine CHEMDIV(4285-2380) CP- 0105772

15. N-(2-(1H- benzo[d]imidazol-2- yl)phenyl)isobutyramide IFLAB/LIFECHEMI- CALS (F0015- 0753)/ CP- 0109953

16. 2-(o-tolyloxymethyl)-1H- benzo[d]imidazole CHEMDIV (6286-0428) CP-0193184

17. 2-(4-methoxyphenyl)-1- phenethyl-1H- benzo[d]imidazole LDDN CP-0064917

18. N-(6- bromobenzo[d]thiazol-2- yl)thiophene-2- carboxamide CHEMDIV(3769-2060) CP- 0067233

19. N-(benzo[d]thiazol-2-yl)- 1-methyl-1H-pyrazole-5- carboxamideCHEMDIV (4487-0569) CP- 0068578

20. 2-(4-fluorobenzylthio) benzo[d]thiazole CHEMDIV (5222-1038) CP-0103014

21. 5-chloro-N- methylbenzo[d]thiazol-2- amine MAY- BRIDGE (RF 04015)CP- 0105777

22. N-(6- acetamidobenzo[d]thiazol- 2-yl)furan-2- carboxamide IFLAB/LIFE- CHEMICALS (F0018- 0056) CP- 0107060

23. N-(6- fluorobenzo[d]thiazol-2- yl)-3-methoxybenzamide IFLAB/LIFe-CHEMICALS (F0412- 0020) CP- 0029300

24. 2-(benzo[d]oxazol-2- ylthio)-N-(2- chlorophenyl)acetamide CHEMDIV(3627-0019) CP- 0079983

25. 5-chloro-2- phenylbenzo[d]oxazole CHEMDIV (K780- 0060) CP- 0103978

26. 5-methyl-2-m- tolylbenzo[d]oxazole MAY- BRIDGE (S 15553) CP- 0034360

27. 2-(4-isobutoxyphenyl)-3- (naphthalen-2-yl)-2,3-dihydroquinazolin-4(1H)- one CHEMDIV (8008-6354) CP- 0036187

28. N-(2-(2-(4-fluorophenyl)- 2-oxoethylthio)-4- oxoquinazolin-3(4H)-yl)benzamide CHEMDIV (K284- 2447) CP- 0050095

29. 2-(4-chlorophenyl)-4-(4- methoxyphenyl)-1,4- dihydrobenzo[4.5]imi-dazo[1,2-a]pyrimidine CHEMDIV (K832- 2696) CP- 0131763

30. 2-(3-pyridyl)-4-(4- bromophenyl)-1,4- dihydrobenzo[4,5]imi-dazo[1,2-a]pyrimidine CHEMDIV (K832- 2426) CP- 0079175

31. N-sec-butyl-1,7,7- trimethyl-9-oxo-8,9- dihydro-7H-furo[3,2-f]chromene-2- carboxamide CHEMDIV (C795- 04798) CP- 0087336

32. N-(3-carbamoyl-5,6- dihydro-4H- cyclopenta[b]thiophen-2-yl)benzofuran-2- carboxamide ENAMINE (T0516- 9815) CP- 0064314

33. 3-chloro-N-(5- chloropyridin-2- yl)benzo[b]thiophene-2- carboxamideCHEMDIV (3616-0520) CP- 0068577

34. 3-chloro-N- ((tetrahydrofuran-2- yl)methyl)benzo[b]thio-phene-2-carboxamide CHEMDIV (5067-0367) CP- 0102404

35. N-(3-(5-chloro-3- methylbenzo[b]thiophen- 2-yl)-1H-pyrazol-5-yl)acetamide MAY- BRIDGE (MWP 00596) CP- 0010539

36. 2-(naphthalen-2-yl)-1H- indole MAY- BRIDGE (RDR 01160) CP- 0072096

37. 2-(pyridin-2-yl)-1H- indole CHEMDIV (8005-4453) CP- 0078448

38. N-(2-chlorophenyl)-2- (1H-indol-3-yl)-2- oxoacetamide CHEMDIV (C730-0133) CP- 0072092

39. 2-m-tolylquinoline CHEMDIV (8005-4434) CP- 0087799

40. 2-(4-(2-methoxyphenyl) piperazin-1-yl)quinoline ENAMINE (T0503-7528) CP- 0009883

41. 2-(1H- benzo[d][1,2,3]triazol-1- yl)-N-(2,3-dihydro-1H-inden-2-yl)acetamide MAY- BRIDGE (KM 10562) CP- 0070871

42. 1-phenethyl-1H- benzo[d][1,2,3]triazole Chem- Bridge (7653692) CP-0063508

43. 7-(4-fluorobenzyloxy)- 2H-chromen-2-one CHEMDIV (3330-4085) CP-0000928

44. N-(2,4-dichlorophenyl)- 8-methoxy-2H- chromene-3-carboxamide BIONET(bionet-5G- 331S) CP- 0005069

45. N-(3-chlorophenyl)-8- methyl-3,4- dihydroquinoline-1(2H)-carbothioamide MAY- BRIDGE (BTB 01026) CP- 0096433

46. 7-methoxy-5-methyl-2- phenyl-4H-chromen-4- one MICRO- SOURCE(01400666) CP- 0045061

47. 2-(3,4-dimethylphenyl) quinoxaline CHEMDIV (3257-1451) CP- 0060729

48. 4-bromo-N-(5- chloropyridin-2- yl)benzamide CHEMDIV (0868-0014) CP-0066751

49. 3-amino-6,7,8,9- tetrahydro-5H- cyclohepta[e]thieno[2,3-b]pyridine-2- carboxamide CHEMDIV (4365-0051) CP- 0069934

50. (Z)-3-methyl-N′- (nicotinoyloxy) benzimidamide CHEMDIV (5906-1071)CP- 0076627

51. N,N-diethyl-6- methoxythieno [2,3-b]quinoline-2- carboxamide CHEMDIV(C303- 0565) CP- 0080276

52. 6-(4-methoxyphenyl)- 1,2,3,4-tetrahydro-1,5- naphthyridine Referencefor Synthesis: Lahue, B. R. et al. J. Org. Chem. 2004, 69, 7171- 7182.CP- 0089966

53. 5-bromo-N-(2- (phenylthio)ethyl) nicotinamide CHEMDIV (8011-8572)CP- 0029278

54. N-(6-methylpyridin-2- yl)-2,3-dihydrobenzo [b][1,4]dioxine-6-carboxamide CHEMDIV (3617-0256) CP- 0130586

55. 2-(4-methylbenzylthio) oxazolo[4,5-b]pyridine CHEMDIV (G293- 0009)CP- 0059547

56. N-(2-methoxyethyl)-5-p- tolylpyrimidin-2-amine PEAK- DALE (1000119)CP- 0059563

57. 4-(5-(benzo[b]thiophen- 2-yl)pyrimidin-2- yl)morpholine PEAK- DALE(1000166) CP- 0059642

58. 4-(5-(4- fluorophenyl)pyrimidin- 2-yl)morpholine PEAKDALE (1000143)CP- 0064382

59. N-(4-bromo-3- methylphenyl) quinazolin-4-amine CHEMDIV (3651-6031)CP- 0067053

60. N-(4-methoxyphenyl) quinazolin-4-amine CHEMDIV (4491-0691) CP-0072720

61. N-(3-methoxyphenyl)- 9H-purin-6-amine CHEMDIV (8009-2985) CP-0079810

62. N,N-diethyl-1-m-tolyl- 1H-pyrazolo[3,4- d]pyrimidin-4-amine CHEMDIV(K402- 0503) or IFLAB/ LIFE- CHEMICALS (F0518- 0004) CP- 0061777

63. (5-(4- bromophenyl)furan-2- yl)(morpholino)methanone CHEMDIV(1975-0198) CP- 0066008

64. (Z)-4-bromo-N′-(furan-2- carbonyloxy)benzimida- mide CHEMDIV(4260-1000) CP- 0072253

65. N-(4-iodophenyl)furan-2- carboxamide CHEMDIV (8002-5214) CP- 0099289

66. 5-(5-(2,4- difluorophenyl)furan-2- yl)-1-(methylsulfonyl)-1H-pyrazole MAY- BRIDGE (CD 10941) CP- 0008545

67. 1-(3-amino-5-(4-tert- butylphenyl)thiophen-2- yl)ethanone MAY-BRIDGE (GK 03407) CP- 0060852

68. N-(3-cyano-4,5,6,7- tetrahydrobenzo[b]thio- phen-2-yl)-2-fluorobenzamide CHEMDIV (1000-0399) CP- 0072156

69. N-(5-chloropyridin-2- yl)thiophene-2- carboxamide CHEMDIV(8005-8364) CP- 0072271

70. N-(2-(4- fluorophenoxy)ethyl)thio- phene-2-carboxamide CHEMDIV(8003-7471) CP- 0104766

71. 2,5-dimethyl-N-phenyl-1- (thiophen-2-ylmethyl)- 1H-pyrrole-3-carboxamide MAY- BRIDGE (SP 00299) CP- 0110352

72. N-(3-cyanothiophen-2- yl)-4- isopropoxybenzamide IFLAB/- LIFE-CHEMI- CALS (F1385- 0110) CP- 0063182

73. 2-(4-methoxyphenoxy)- N-(thiazol-2- yl)acetamide CHEMDIV (3297-0008)CP- 0071862

74. 4-(4-methoxyphenyl)-N- (3-methylpyridin-2- yl)thiazol-2-amineCHEMDIV (7100-0567) CP- 0072036

75. 4-(biphenyl-4-yl) thiazol-2-amine CHEMDIV (8005-3411) CP- 0105343

76. 4-(4-(4- methoxyphenyl)thiazol- 2-yl)-3-methylisoxazol-5- amine MAY-BRIDGE (SPB 05463) CP- 0122949

77. N-(2-methoxyphenyl)-4- phenylthiazol-2-amine CHEMDIV (0896-3691) CP-0134381

78. 1-(4-amino-2-(m- tolylamino)thiazol-5-yl)- 2-methylpropan-1-oneCHEMDIV (F091-0329) CP- 0000477

79. 4-(4-chlorophenyl)-1- (5H-pyrimido[5,4- b]indol-4-yl)-1H-pyrazol-3-amine BIONET (bionet-5F- 909) CP- 0063375

80. 2-(4-chlorophenyl)-6- ethyl-5- methylpyrazolo[1,5-a]pyrimidin-7(4H)-one CHEMDIV (3270-0084) CP- 0064231

81. 5-methoxy-2-(5-phenyl- 1H-pyrazol-3-yl)phenol CHEMDIV (3486-0181)CP- 0065105

82. (3-(4-bromophenyl)-1- phenyl-1H-pyrazol-4- yl)methanol CHEMDIV(3935-0218) CP- 0070844

83. N-(2,5-dichlorophenyl)- 1-ethyl-1H-pyrazole-3- carboxamide CHEMDIV(6228-1918) CP- 0070886

84. 4-chloro-1-methyl-N-(2- oxo-2-phenylethyl)-1H-pyrazole-3-carboxamide Chem- Bridge (7528295) CP- 0104765

85. N-(3-(5-tert-butyl-2- methylfuran-3-yl)-1H- pyrazol-5-yl)benzamideMAY- BRIDGE (SP 00221) CP- 0065665

86. N-(5-methylisoxazol-3- yl)benzo[d][1,3]dioxole- 5-carboxamideCHEMDIV (4100-3780) CP- 0075627

87. (5-(4- bromophenyl)isoxazol-3- yl)(morpholino)methanone CHEMDIV(C226- 0488) CP- 0075656

88. N-(4-bromophenyl)-5- isopropylisoxazole-3- carboxmaide CHEMDIV(C226-292) CP- 0067108

89. 5-((4-chloro-2- methylphenoxy)methyl)- 3-(pyridin-4-yl)-1,2,4-oxadiazole CHEMDIV (4534-3904) CP- 0067246

90. 5-(2-methoxyphenyl)-3- p-tolyl-1,2,4-oxadiazole CHEMDIV (4534-1114)CP- 0068395

91. 5-(phenoxymethyl)-3- (pyridin-2-yl)-1,2,4- oxadiazole CHEMDIV(4951-0941) CP- 0068929

92. 5-(2-chloro-4- methylphenyl)-3- (pyridin-3-yl)-1,2,4- oxadiazoleCHEMDIV (5235-0410) CP- 0068961

93. 3-(2-chlorophenyl)-5-p- tolyl-1,2,4-oxadiazole CHEMDIV (5235-2061)CP- 0070164

94. 5-(piperidin-1-ylmethyl)- 3-p-tolyl-1,2,4- oxadiazole CHEMDIV(5927-0188) CP- 0070367

95. 5-(4-bromophenyl)-3- (pyridin-3-yl)-1,2,4- oxadiazole CHEMDIV(6018-0130) CP- 0079642

96. 5-(2-bromophenyl)-3-(4- bromophenyl)-1,2,4- oxadiazole CHEMDIV(K086- 0188) CP- 0104904

97. 5-(2-bromo-5- methoxyphenyl)-3- (thiophen-2-yl)-1,2,4- oxadiazoleMAY- BRIDGE (SP-00905) CP- 0130665

98. 3-(2-fluorophenyl)-N-(3- (piperidin-1-yl)propyl)-1,2,4-oxadiazol-5-amine CHEMDIV (G349- 0769) CP- 0005186

99. 2-(2-chlorobenzoyl)-N- (4- fluorophenyl)hydrazine- carbothioamideMAY- BRIDGE (BTB 01235) CP- 0007991

100. 2-(methylamino)-N- phenethylbenzamide MAY- BRIDGE (DP 01029) CP-0061566

101. 4-tert-butyl-N- ((tetrahydrofuran-2- yl)methyl)benzamide CHEMDIV(1786-0077) CP- 0062030

102. 2-phenyl-5-o-tolyl-1,3,4- oxadiazole CHEMDIV (2089-0007) CP-0007994

103. 4-(3-(4-chlorophenyl)- 4,5-dihydro-1H-1,2,4- triazol-5-yl)-N,N-dimethylaniline MAY- BRIDGE (DP 01118) CP- 0039073

104. 7-methoxy-2-(4- methoxyphenyl)-1,10b- dihydrospiro[benzo[e]pyra-zolo[1,5-c][1,3]oxazine- 5,1′-cyclohexane] CHEMDIV (K805- 0823) CP-0004116

105. 6-oxo-2-(4-(3- (trifluoromethyl)phenoxy) phenyl)-1,4,5,6-tetrahydropyridine-3- carbonitrile BIONET (bionet-8P- 057) CP- 0061401

106. 6-(4-methoxyphenyl) imidazo[2,1-b]thiazole CHEMDIV (1487-1266) CP-0064286

107. 2-(2-bromophenoxy)-N- (4H-1,2,4-triazol-3- yl)acetamide CHEMDIV(3643-3466) CP- 0110644

108. 1-(indolin-1-yl)-2- phenoxyethanone TIMTEC (ST040751) CP- 0051092

109. 2-(4-chlorophenyl)- 6,7,8,9- tetrahydrobenzo[e]imida-zo[1,2-b][1,2,4]triazine CHEMDIV (8011-7131) ^(A)The systematic namesprovided in Table 1 were generated using ChemDraw Ultra Version 9.0.1software as follows. The systematic names were generated by inputtingeach of the chemical structures shown in Table 1 in the ChemDraw drawingwindow, selecting the compound, and selecting the “convert structure toname” tool under the Structure menu.Methods of Treatment

The present invention provides methods related to the use of thecompounds described herein for treating diseases and/or disorders thatwould benefit from increased Atoh1 expression. In general, the methodsof treatment involve the use of one or more of the compounds describedherein to increase Atoh1 expression levels, and thereby promote partialor complete differentiation of a target cell. Diseases that can benefitfrom such treatment are those in which increased levels of Atoh1 treatone or more symptoms of the disease, e.g., those diseases in which thecompletely or partially differentiated cells that result from increasedAtoh1 expression (1) serve to replace lost or damaged cells or tissue,e.g., functional cells, e.g., auditory hair cells, and/or (2) preventexpansion of a damaging population of cells, e.g., cancer cells.

In general, the present invention provides steps whereby one or more ofthe compounds described herein are administered to a patient.Alternatively or in addition, the present invention provides stepswhereby one or more target cells e.g., stem cells, iPS cells, progenitorcells, and/or support cells are contacted, e.g., in vitro, with one ormore of the compounds described herein to promote complete or partialdifferentiation of those cells to or toward a mature cell type, e.g., ahair cell, steps whereby one or more cells, e.g., cells, progenitorcells, and/or support cells that have been contacted with one or more ofthe compounds described herein, e.g., in vitro, is administered to apatient, and/or steps whereby one or more cells, e.g., cells, progenitorcells, and/or support cells that have been contacted with one or more ofthe compounds described herein, e.g., in vitro are administered to apatient in combination with one or more of the compounds.

Auditory Hair Cell Loss

It is widely accepted that although cells capable of generating haircells are present in the inner ear, natural hair cell regeneration inthe inner ear is low (Li et al., Trends Mol. Med., 10, 309-315 (2004);Li et al., Nat. Med., 9, 1293-1299 (2003); Rask-Andersen et al., Hear.Res., 203, 180-191 (2005)). As a result, lost or damaged hair cells maynot be adequately replaced by natural physiological processes (e.g.,cell differentiation) and a loss of hair cells occurs. In manyindividuals, such hair cell loss can result in, e.g., sensorineuralhearing loss, hearing impairment, and imbalance disorders. Therapeuticstrategies that increase the number of hair cells in the inner ear willbenefit a patient with hair cell loss, e.g., with one or more of theseconditions.

The importance of Atoh1 in hair cell genesis is well documented. Forexample, Math1 is required for hair cell development and thedifferentiation of inner ear progenitor cells to inner ear support cellsand/or hair cells (Bermingham et al., Science, 284:1837-1841, 1999). Inaddition, adenovirus mediated Math1 overexpression in the endolymph ofthe mature guinea pig results in the differentiation of non-sensorycells in the mature cochlea into immature hair cells (Kawamoto et al.,J. Neurosci., 23:4395-4400, 2003). The implications of these studies aretwofold. First, they demonstrate that non-sensory cells of the maturecochlear retain the ability to differentiate into sensory cells, e.g.,hair cells. Second, they demonstrate that Math1 overexpression isnecessary and sufficient to direct hair cell differentiation fromnon-sensory cells. A later study furthered these findings bydemonstrating that adenovirus mediated Atoh1 overexpression induces haircell regeneration and substantially improves hearing thresholds in anexperimentally deafened animal model (Izumikawa et al., Nat. Med.,11:271-276, 2005).

Provided herein are compounds capable of increasing Atoh1 levels in asubject and/or cell or tissue. As described herein, these compoundspromote increased Atoh1 expression and thereby promote differentiationof a target cell or cells to or toward sensory cell or cells of theinner ear, e.g., a hair cell. The use of these compounds to promote haircell differentiation from cells located in the ear, or from cellscapable of differentiating into a hair cell is well supported at leastby the experimental data described by Bermingham et al., supra, Kawamotoet al., supra, and Izumikawa et al., supra. Consequently, the compoundsdescribed herein can be used to treat those diseases and disorders thatresult from hair cell loss in a patient.

The present invention provides compounds and methods for treatingpatients who have, or who are at risk for developing, an auditorydisorder resulting from a loss of hair cells. In some embodiments, themethods of treatment include steps whereby one or more of the compoundsdescribed herein are administered to a patient to promote the formationof auditory hair cells, e.g., in the ear of the patient (e.g., the innerear) and/or increase the number of auditory hair cells in the ear (e.g.,the inner ear) of a patient by promoting complete or partial auditoryhair cell differentiation from non-hair cell types naturally present inthe inner ear of a patient.

In some embodiments, the methods of treatment include steps whereby oneor more of the compounds described herein are administered to a patientto promote the formation of auditory hair cells in the patient's innerear (e.g., an inner and/or outer auditory hair cells) and/or increasethe number of auditory hair cells (e.g., an inner and/or outer auditoryhair cells) in the inner ear of a patient by promoting complete orpartial auditory hair cell differentiation from non-hair cell typesnaturally present in the inner ear of a patient.

Examples of cells that are capable of differentiating into hair cells(e.g., an inner and/or outer hair cells) include but are not limited toinner ear stem cells, iPS cells, progenitor cells, and/or support cells(e.g., Deiters' cells, pillar cells, inner phalangeal cells, tectalcells and Hensen's cells).

The present invention also include steps whereby one or more cells thatare capable of differentiating completely or partially into a hair cellare contacted, e.g., in vitro, with one or more of the compoundsdescribed herein to promote complete or partial differentiation of thosecells to or toward a mature cell type of the inner ear, e.g., a haircell (e.g., an inner and/or outer hair cell). Exemplary cells that arecapable of differentiating into a hair cell include, but are not limitedto stem cells (e.g., inner ear stem cells, adult stem cells, bone marrowderived stem cells, embryonic stem cells, mesenchymal stem cells, skinstem cells, iPS cells, and fat derived stem cells), progenitor cells(e.g., inner ear progenitor cells), support cells (e.g., Deiters' cells,pillar cells, inner phalangeal cells, tectal cells and Hensen's cells),and/or germ cells.

Alternatively or in addition, the methods include steps whereby one ormore cells that are capable of differentiating into a hair cell (e.g.,an inner and/or outer hair cell) and that have been contacted with oneor more of the compounds described herein, e.g., in vitro, areadministered to the ear (e.g., the inner ear) of the patient (celltherapy). Finally, the methods include steps whereby one or more cellsthat are capable of differentiating into a hair cell (e.g., an innerand/or outer hair cell) and that have been contacted with one or more ofthe compounds described herein, e.g., in vitro are administered to theear (e.g., inner ear) of a patient in combination with one or more ofthe compounds (combination therapy).

The present invention can be used to treat hair cell loss and anydisorder that arises as a consequence of cell loss in the ear, such ashearing impairments, deafness, and vestibular disorders, for example, bypromoting differentiation (e.g., complete or partial differentiation) ofone or more cells into one or more cells capable of functioning assensory cells of the ear, e.g., hair cells.

In some embodiments, the methods include steps of selecting a patient atrisk of hair cell loss and/or a patient with hair cell loss.Alternatively or in addition, the methods include steps of selecting apatient at risk of sensorineural hearing loss and/or a patient withsensorineural hearing loss. For example, any human experiencing or atrisk for developing hearing loss is a candidate for the treatmentmethods described herein. A human having or at risk for developing ahearing loss can hear less well than the average human being, or lesswell than a human before experiencing the hearing loss. For example,hearing can be diminished by at least 5, 10, 30, 50% or more.

The subject can a hearing loss associated with hair cell loss for anyreason, or as a result of any type of event. For example, a human can bedeaf because of a genetic or congenital defect; for example, a human canhave been deaf since birth, or can be deaf or hard-of-hearing as aresult of a gradual loss of hearing due to a genetic or congenitaldefect. In another example, a human can be deaf or hard-of-hearing as aresult of a traumatic event, such as a physical trauma to a structure ofthe ear, or a sudden loud noise, or a prolonged exposure to loud noises.For example, prolonged exposures to concert venues, airport runways, andconstruction areas can cause inner ear damage and subsequent hearingloss. A human can experience chemical-induced ototoxicity, whereinototoxins include therapeutic drugs including antineoplastic agents,salicylates, quinines, and aminoglycoside antibiotics, contaminants infoods or medicinals, and environmental or industrial pollutants. A humancan have a hearing disorder that results from aging, or the human canhave tinnitus (characterized by ringing in the ears).

A human suitable for the treatment using the compounds and methodsfeatured in the invention can include a human having a vestibulardysfunction, including bilateral and unilateral vestibular dysfunction.Vestibular dysfunction is an inner ear dysfunction characterized bysymptoms that include dizziness, imbalance, vertigo, nausea, and fuzzyvision and may be accompanied by hearing problems, fatigue and changesin cognitive functioning. Vestibular dysfunction can be the result of agenetic or congenital defect; an infection, such as a viral or bacterialinfection; or an injury, such as a traumatic or nontraumatic injury.Vestibular dysfunction is most commonly tested by measuring individualsymptoms of the disorder (e.g., vertigo, nausea, and fuzzy vision).

Alternatively or in addition, the compounds and methods featured in theinvention can be used prophylactically, such as to prevent, reduce ordelay progression of hearing loss, deafness, or other auditory disordersassociated with loss of inner ear function. For example, a compositioncontaining one or more compounds can be administered with (e.g., before,after or concurrently with) a second therapeutic, such as a therapeuticthat may affect a hearing disorder. Such ototoxic drugs include theantibiotics neomycin, kanamycin, amikacin, viomycin, gentamycin,tobramycin, erythromycin, vancomycin, and streptomycin;chemotherapeutics such as cisplatin; nonsteroidal anti-inflammatorydrugs (NSAIDs) such as choline magnesium trisalicylate, diclofenac,diflunisal, fenoprofen, flurbiprofen, ibuprofen, indomethacin,ketoprofen, meclofenamate, nabumetone, naproxen, oxaprozin,phenylbutazone, piroxicam, salsalate, sulindac, and tolmetin; diuretics;salicylates such as aspirin; and certain malaria treatments such asquinine and chloroquine. For example, a human undergoing chemotherapycan be treated using the compounds and methods described herein. Thechemotherapeutic agent cisplatin, for example, is known to cause hearingloss. Therefore, a composition containing one or more compounds can beadministered with cisplatin therapy (e.g., before, after or concurrentlywith) to prevent or lessen the severity of the cisplatin side effect.Such a composition can be administered before, after and/orsimultaneously with the second therapeutic agent. The two agents may beadministered by different routes of administration.

The compounds and methods featured in the invention are appropriate forthe treatment of hearing disorders resulting from sensorineural haircell loss. Patients with sensorineural hair cell loss experience thedegeneration of cochlear hair cells, which frequently results in theloss of spiral ganglion neurons in regions of hair cell loss. Suchpatients may also experience loss of supporting cells in the organ ofCorti, and degeneration of the limbus, spiral ligament, and striavascularis in the temporal bone material. These patients can receivetreatment with an agent that causes cells to differentiate into haircells, or a tissue transplant containing hair cells grafted or injectedinto the inner ear.

Methods of generating cells of the inner ear are provided below. Earcells or ear cell progenitors can be generated from stem cells isolatedfrom a mammal, such as a mouse or human, and the cells can be embryonicstem cells or stem cells derived from mature (e.g., adult) tissue, suchas the inner ear, central nervous system, blood, skin, eye or bonemarrow. Any of the methods described below for culturing stem cells andinducing differentiation into ear cells (e.g., hair cells) can be used.

In general, the compounds and methods described herein can be used togenerate hair cell growth in the ear and/or to increase the number ofhair cells in the ear (e.g., in the inner, middle, and/or outer ear).For example, the number of hair cells in the ear can be increased about2-, 3-, 4-, 6-, 8-, or 10-fold, or more, as compared to the number ofhair cells before treatment. This new hair cell growth can effectivelyrestore or establish at least a partial improvement in the subject'sability to hear. For example, administration of an agent can improvehearing loss by about 5, 10, 15, 20, 40, 60, 80, 100% or more.

Where appropriate, following treatment, the human can be tested for animprovement in hearing or in other symptoms related to inner eardisorders. Methods for measuring hearing are well-known and include puretone audiometry, air conduction, and bone conduction tests. These examsmeasure the limits of loudness (intensity) and pitch (frequency) that ahuman can hear. Hearing tests in humans include behavioral observationaudiometry (for infants to seven months), visual reinforcementorientation audiometry (for children 7 months to 3 years) and playaudiometry for children older than 3 years. Oto-acoustic emissiontesting can be used to test the functioning of the cochlear hair cells,and electro-cochleography provides information about the functioning ofthe cochlea and the first part of the nerve pathway to the brain. Insome embodiments, treatment can be continued with or withoutmodification or can be stopped.

Abnormal Cell Proliferation

Cell proliferation is normally a tightly regulated process that isgoverned by multiple checkpoints and safeguards. Abnormal cellproliferation occurs when one or more of these checkpoints or safeguardsare bypassed or breakdown, e.g., through genetic mutation. The result ofabnormal cell proliferation is the formation of cancerous growths ortumors. The most aggressive cancerous growths are typically invasive andmetastatic. Less aggressive benign growths are not invasive ormetastatic, although they frequently retain the potential to becomemetastatic.

In some embodiments, the present invention is directed to methods andcompositions for the treatment of abnormal cell proliferation and/orcancer, which would benefit from increased Atoh1 expression. Abnormallyproliferating or cancerous cells that can benefit from increased Atoh1expression can be identified by determining Atoh1 expression levelswithin the cells, e.g., using real-time PCR and other techniques thatcan be readily performed by one of skill in the art. Such determinationscan be performed by obtaining a sample of abnormally proliferating orcancerous cells from a subject; isolating the genetic material from thesample (e.g., DNA and RNA); reverse transcribing the mRNA from thesample; and amplifying a Atoh1 sequence using oligonucleotides that havebeen designed to hybridize to a Atoh1 sequence. Abnormally proliferatingcells or cancer cells that can benefit from increased Atoh1 expressionwill have undetectable Atoh1 expression in the cell sample.Alternatively or in addition, the above determination will be repeatedusing a non-cancerous cell control. Atoh1 expression in the control willthen be compared to Atoh1 expression in the cancerous sample. Abnormallyproliferating cells or cancer cells that can benefit from increasedAtoh1 expression will have less Atoh1 expression than the non-cancerouscontrol. In general, abnormally proliferating cells or cancer cells thatcan benefit from increased Atoh1 expression will have low orundetectable Atoh1 expression levels.

In some embodiments, the present invention is directed to methods andcompositions for the treatment of abnormal cell proliferation and/orcancer in the gastrointestinal system. Exemplary cancers include but arenot limited to cancers of the esophagus, gallbladder, liver, pancreas,stomach, small intestine, large intestine (colon) and rectum.

Support for the use of the present invention for the treatment ofabnormal cell proliferation and/or cancer of the gastrointestinal systemis provided by the following studies: Normally, the intestinalepithelium consists of four main cell types that derived from onemultipotent stem cell during embryogenesis. The first cell type is theabsorptive enterocyte or columnar cell; the second cell type is themucous secreting goblet cell; the third cell type is the regulatorypeptide-secreting enteroendocrine cell; and the fourth cell type is theantimicrobial peptide-secreting Paneth cell. A healthy animal will haveeach of these four cell types. Math1 null transgenic mice, however, havedepleted goblet, enteroendocrine, and paneth cells. This observation haslead to the conclusion that Math1 is required for cell fatedetermination (e.g., differentiation) towards these three cell types inthe developing gut (Yang et al., Science, 294:2155-2158, 2001). It hasalso been demonstrated that Hath1 expression is absent in five gastriccancer cell lines compared with normal gastric mucosae. This supportsthe fact that the loss of Hath1 expression may play a role in gastriccarcinogenesis (Sekine et al., Biochem. Biophys., Res. Comm.,344:1166-1171, 2006). Decreased Hath1 and Math1 expression in coloncancer cell lines is also reported elsewhere (Leow et al., Cancer Res.,64:6050-6057, 2004 and Leow et al., Ann. N.Y. Acad. Sci., 1059:174-183,2005). These studies, however, also demonstrate that Hath1overexpression in an aggressive colon cancer cell line results in asignificant inhibition of cell proliferation, and that this decreasedproliferation occurs because the aggressive colon cancer cellsdifferentiate to or towards goblet cells, which are not cancerous. Thesedata, therefore, clearly suggest that gastrointestinal cancer willbenefit from increased Atoh1 expression, for example, by reducing thenumber of proliferating gastric cancer cells by promoting thedifferentiation of such cells to or towards a non-cancerous cell of theintestinal epithelium. Consequently, a patient with gastrointestinalcancer can be treated with one or more of the compounds describedherein.

In general, the present invention provides compounds and methods fortreating patients who have, or who are at risk for developinggastrointestinal cancer. Methods for identifying such a patient aredescribed below. The methods of treatment include steps whereby one ormore of the compounds described herein are administered to a patient totreat gastrointestinal cancer (direct therapy).

In some embodiments, the methods include methods of selecting a patientat risk of gastrointestinal cancer and/or a patient withgastrointestinal cancer.

Methods for identifying a patient with gastrointestinal cancer are knownin the art. For example, screens can include the use of endoscopy (e.g.,oral and/or rectal). Screens can also include tests to detect variousimmunohistochemical markers, including but not limited to, e.g., CK20,MUC2, MUC5A, MUC6, DAS-1, and CDX2.

The present invention is useful for providing treatment for patients whohave or who are at risk for developing gastrointestinal cancer using oneor more of the compounds described herein. The methods of treatmentinclude steps whereby one or more of the compounds described herein areadministered to a patient to promote complete or partial differentiationof gastric cancer cells.

In some embodiments, the present invention is directed to methods andcompositions for the treatment of colorectal cancer. Screens foridentifying individuals with colorectal cancer are known in the art. Forexample, screens for colorectal cancer include: fecal occult blood test(FOBT), which checks for blood in the stool, digital rectal exam (DRE),which checks for tactile abnormalities in the rectum, sigmoidoscopy,which looks for visual abnormality in the rectum and lower part of thecolon, colonoscopy, which allows visualization of the rectum and entirecolon, and double contrast barium enema (DCBE), which allowsradiographic examination of the rectum and colon. Frequently, a biopsyor polypectomy of abnormal colorectal tissue is examined to confirm thatthe tissue is cancerous.

Individuals with colorectal cancer can be classified according to cancerstage scales, such as the Dukes, Astler-Coller, and AJCC/TNM scales. Anindividual's grade of cancer indicates the degree of de-differentiationthe cancer cells have undergone, i.e., how much the tumor's cells stillretain the characteristics of a colon or rectal cell. Stage groupingsare indicative of person's overall disease stage. In some systems, stagegroupings are expressed as Roman numerals from 0 (the earliest stage) toIV (the most advanced stage). In stage 0, the cancer is found only inthe inner lining of the colon or rectum. In stage I, the cancer hasspread to more of the inner wall of the colon or rectum. In stage II,the cancer has spread outside the colon or rectum to nearby tissue, buthas not spread to the lymph nodes. In stage III, the cancer has spreadto nearby lymph nodes but not to other parts of the body. In stage IV,the cancer has spread to other parts of the body. Colorectal cancertends to spread to the liver and/or lungs. (Stages 0 and IV, justdescribed, correspond to stages A and D, respectively, in the Dukescale). Further information on the screening, diagnosis, and staging ofcolorectal cancer can be found in Frei et al., Cancer Medicine, BCDecker Inc., Hamilton, Ontario (2003).

The present invention is useful for providing treatment for patients whohave (e.g., stages 0 to IV), or who are at risk for developing,colorectal cancer using one or more of the compounds described herein.The methods of treatment include steps whereby one or more of thecompounds described herein are administered to a patient to promotecomplete or partial differentiation of colorectal cancer cells.

In some embodiments, a patient undergoing treatment or having completedtreatment for colon cancer may be reevaluated, e.g., using the methodsdescribed above, to determine the effectiveness of therapy. In someembodiments, treatment can be continued with or without modification orcan be stopped.

Other Conditions

Atoh1 expression is also reported in the cerebellum and dorsal spinalcord and has an important role, e.g., in development (Bermingham et al.,supra and Helms et al., supra). Atoh1 clearly has a role in promotingcell differentiation in neural cells and tissues beyond those found inthe inner ear. The compounds and pharmaceutical compositions describedherein, therefore, can also be used for the treatment of diseases and/ordisorders of such tissues that would benefit from increased Atoh1expression.

Alternatively or in addition, the present invention can be used to treatcerebellar granule neuron deficiencies, joint disease, andosteoarthritis.

Conditions that can benefit from increased Atoh1 expression can beidentified by determining Atoh1 expression levels within a cell using,e.g., the RT-PCR methods described above. In general, conditions thatcan benefit from increased Atoh1 expression will have low orundetectable Atoh1 expression levels.

Routes of Administration for the Treatment of Auditory Hair Cell Loss

Direct Therapy

The route of administration will vary depending on the disease beingtreated. Hair cell loss and/or sensorineural hearing loss can be treatedusing direct therapy using systemic administration and/or localadministration. In some embodiments, the route of administration can bedetermined by a patient's health care provider or clinician, for examplefollowing an evaluation of the patient. In some embodiments, aindividual patient's therapy may be customized, e.g., one or morecompounds, the routes of administration, and the frequency ofadministration can be personalized. Alternatively, therapy may beperformed using a standard course of treatment, e.g., using one or morepre-selected compounds and pre-selected routes of administration andfrequency of administration.

In some embodiments, one or more of the compounds described herein canbe administered to a patient, e.g., a patient identified as being inneed of treatment for hair cell loss, using a systemic route ofadministration. Systemic routes of administration can include, but arenot limited to, parenteral routes of administration, e.g., intravenousinjection, intramuscular injection, and intraperitoneal injection;enteral routes of administration e.g., administration by the oral route,lozenges, compressed tablets, pills, tablets, capsules, drops (e.g., eardrops), syrups, suspensions and emulsions; rectal administration, e.g.,a rectal suppository or enema; a vaginal suppository; a urethralsuppository; transdermal routes of administration; and inhalation (e.g.,nasal sprays).

Alternatively or in addition, one or more of the compounds describedherein can be administered to a patient, e.g., a patient identified asbeing in need of treatment for hair cell loss, using a local route ofadministration. Such local routes of administration includeadministering one or more of the compounds described herein into the earof a patient and/or the inner ear of a patient, for example, byinjection and/or using a pump.

In some embodiments, a pharmaceutical composition can be injected intothe ear (e.g., auricular administration), such as into the luminae ofthe cochlea (e.g., the Scala media, Sc vestibulae, and Sc tympani),e.g., using a syringe, e.g., a single-dose syringe. For example, one ormore of the compounds described herein can be administered byintratympanic injection (e.g., into the middle ear), and/or injectionsinto the outer, middle, and/or inner ear. Such methods are routinelyused in the art, for example, for the administration of steroids andantibiotics into human ears. Injection can be, for example, through theround window of the ear or through the cochlear capsule. Other inner earadministration methods are known in the art (see, e.g., Salt andPlontke, Drug Discovery Today, 10:1299-1306, 2005).

In another mode of administration, the pharmaceutical composition can beadministered in situ, via a catheter or pump. A catheter or pump can,for example, direct a pharmaceutical composition into the cochlearluminae or the round window of the ear and/or the lumen of the colon.Exemplary drug delivery apparatus and methods suitable for administeringone or more of the compounds described herein into an ear, e.g., a humanear, are described by McKenna et al., (U.S. Publication No.2006/0030837) and Jacobsen et al., (U.S. Pat. No. 7,206,639). In someembodiments, a catheter or pump can be positioned, e.g., in the ear(e.g., the outer, middle, and/or inner ear) of a patient during asurgical procedure. In some embodiments, a catheter or pump can bepositioned, e.g., in the ear (e.g., the outer, middle, and/or inner ear)of a patient without the need for a surgical procedure.

Alternatively or in addition, one or more of the compounds describedherein can be administered in combination with a mechanical device suchas a cochlear implant or a hearing aid, which is worn in the outer ear.An exemplary cochlear implant that is suitable for use with the presentinvention is described by Edge et al., (U.S. Publication No.2007/0093878).

In some embodiments, the modes of administration described above may becombined in any order and can be simultaneous or interspersed.

Alternatively or in addition, the present invention may be administeredaccording to any of the Food and Drug Administration approved methods,for example, as described in CDER Data Standards Manual, version number004 (which is available at fda.give/cder/dsm/DRG/drg00301.htm).

Cell Therapy

In general, the cell therapy methods described herein can be used topromote complete or partial differentiation of a cell to or towards amature cell type of the inner ear (e.g., a hair cell) in vitro. Cellsresulting from such methods can then be transplanted or implanted into apatient in need of such treatment. The cell culture methods required topractice these methods, including methods for identifying and selectingsuitable cell types, methods for promoting complete or partialdifferentiation of selected cells, methods for identifying complete orpartially differentiated cell types, and methods for implanting completeor partially differentiated cells are described below.

Cell Selection

Cells suitable for use in the present invention include, but are notlimited to, cells that are capable of differentiating completely orpartially into a mature cell of the inner ear, e.g., a hair cell (e.g.,an inner and/or outer hair cell), when contacted, e.g., in vitro, withone or more of the compounds described herein. Exemplary cells that arecapable of differentiating into a hair cell include, but are not limitedto stem cells (e.g., inner ear stem cells, adult stem cells, bone marrowderived stem cells, embryonic stem cells, mesenchymal stem cells, skinstem cells, iPS cells, and fat derived stem cells), progenitor cells(e.g., inner ear progenitor cells), support cells (e.g., Deiters' cells,pillar cells, inner phalangeal cells, tectal cells and Hensen's cells),and/or germ cells. The use of stem cells for the replacement of innerear sensory cells is described in Li et al., (U.S. Publication No.2005/0287127) and Li et al., (U.S. patent Ser. No. 11/953,797). The useof bone marrow derived stem cells for the replacement of inner earsensory cells is described in Edge et al., PCT/US2007/084654. iPS cellsare described, e.g., at Takahashi et al., Cell, Volume 131, Issue 5,Pages 861-872 (2007); Takahashi and Yamanaka, Cell 126, 663-76 (2006);Okita et al., Nature 448, 260-262 (2007); Yu, J. et al., Science318(5858):1917-1920 (2007); Nakagawa et al., Nat. Biotechnol. 26:101-106(2008); and Zaehres and Schöler, Cell 131(5):834-835 (2007).

Such suitable cells can be identified by analyzing (e.g., qualitativelyor quantitatively) the presence of one or more tissue specific genes.For example, gene expression can be detected by detecting the proteinproduct of one or more tissue-specific genes. Protein detectiontechniques involve staining proteins (e.g., using cell extracts or wholecells) using antibodies against the appropriate antigen. In this case,the appropriate antigen is the protein product of the tissue-specificgene expression. Although, in principle, a first antibody (i.e., theantibody that binds the antigen) can be labeled, it is more common (andimproves the visualization) to use a second antibody directed againstthe first (e.g., an anti-IgG). This second antibody is conjugated eitherwith fluorochromes, or appropriate enzymes for colorimetric reactions,or gold beads (for electron microscopy), or with the biotin-avidinsystem, so that the location of the primary antibody, and thus theantigen, can be recognized.

Tissue-specific gene expression can also be assayed by detection of RNAtranscribed from the gene. RNA detection methods include reversetranscription coupled to polymerase chain reaction (RT-PCR), Northernblot analysis, and RNAse protection assays.

Exemplary tissue specific genes that may be used to identify a stem cell(e.g., an undifferentiated cell) include, but are not limited to, e.g.,nestin, sox1, sox2, or musashi, NeuroD, Atoh1, and neurogenin1.Alternatively or in addition, stem cells can be selected based on one ormore of the unique properties that such cell types present in vitro. Forexample, in vitro, stem cells often show a distinct potential forforming spheres by proliferation of single cells. Thus, theidentification and isolation of spheres can aid in the process ofisolating stem cells from mature tissue for use in making differentiatedcells of the inner ear. For example, stem cells can be cultured in serumfree DMEM/high-glucose and F12 media (mixed 1:1), and supplemented withN2 and B27 solutions and growth factors. Growth factors such as EGF,IGF-1, and bFGF have been demonstrated to augment sphere formation inculture.

Exemplary tissue specific genes that may be used to identify aprogenitor cells and/or an inner ear progenitor cell (e.g., a less thanfully differentiated or partially differentiated cell) include but arenot limited to, e.g., nestin, sox2, and musashi, in addition to certaininner-ear specific marker genes such as Brn3c, islet1 and Pax2

Exemplary tissue specific genes that may be used to identify fullydifferentiated cells (e.g., support cells) include, but are not limitedto, e.g., p27_(kip), p75, S100A, Jagged-1, and Prox 1.

Exemplary tissue specific genes that may be used to identify fullydifferentiated cells capable of functioning as inner ear sensory cells)include, but are not limited to, e.g., myosin VIIa, Math1, α9acetylcholine receptor, espin, parvalbumin 3, and F-actin (phalloidin).

Alternatively or in addition, cells suspected as being fullydifferentiated (e.g., cells capable of functioning as inner ear sensorycells) may be subjected to physiological testing to determine whetherconductance channels that would be present in mature hair cells arepresent and active.

Alternatively or in addition, inner ear hair cells may be distinguishedfrom other fully differentiated cells of the inner ear (e.g., spiralganglia) by analyzing the expression of markers that are specific tospiral ganglia, which include but are not limited to ephrinB2, ephrinB3,trkB, trkC, GATA3, and BF1.

In some embodiments, suitable cells can be derived from a mammal, suchas a human, mouse, rat, pig, sheep, goat, or non-human primate. Forexample, stem cells have been identified and isolated from the mouseutricular macula (Li et al., Nature Medicine 9:1293-1299, 2003). Thecells can also be obtained from a patient to whom they will subsequentlybe re-administered.

In some embodiments, suitable cells (e.g., a stem cell, progenitor cell,and/or support cell) may be isolated from the inner ear of an animal.More specifically, a suitable cells can be obtained from the cochlearorgan of Corti, the modiolus (center) of the cochlea, the spiralganglion of the cochlea, the vestibular sensory epithelia of thesaccular macula, the utricular macula, or the cristae of thesemicircular canals. The stem cell, progenitor cell, and/or supportcells can also be obtained, however, from other tissues such as bonemarrow, blood, skin, or an eye. The cells employed can be obtained froma single source (e.g., the ear or a structure or tissue within the ear)or a combination of sources (e.g., the ear and one or more peripheraltissues (e.g., bone marrow, blood, skin, or an eye)).

Alternatively or in addition, methods include obtaining tissue from theinner ear of the animal, where the tissue includes at least a portion ofthe utricular maculae. The animal can be a mammal, such as a mouse, rat,pig, rabbit, goat, horse, cow, dog, cat, primate, or human. The isolatedtissue can be suspended in a neutral buffer, such as phosphate bufferedsaline (PBS), and subsequently exposed to a tissue-digesting enzyme(e.g., trypsin, leupeptin, chymotrypsin, and the like) or a combinationof enzymes, or a mechanical (e.g., physical) force, such as trituration,to break the tissue into smaller pieces. Alternatively, or in addition,both mechanisms of tissue disruption can be used. For example, thetissue can be incubated in about 0.05% enzyme (e.g., about 0.001%,0.01%, 0.03%, 0.07%, or 1.0% of enzyme) for about 5, 10, 15, 20, or 30minutes, and following incubation, the cells can be mechanicallydisrupted. The disrupted tissue can be passed through a device, such asa filter or bore pipette, that separates a stem cell or progenitor cellfrom a differentiated cell or cellular debris. The separation of thecells can include the passage of cells through a series of filtershaving progressively smaller pore size. For example, the filter poresize can range from about 80 μm or less, about 70 μm or less, about 60μm or less, about 50 μm or less, about 40 μm or less, about 30 μm orless, about 35 μm or less, or about 20 μm or less.

The cells obtained may constitute an enriched population of stem cellsand/or progenitor cells; isolation from all (or essentially all)differentiated cells or other cellular material within the tissue may beachieved but is not required to meet the definition of “isolated.”Absolute purity is not required. The invention encompasses cellsobtained by the isolation procedures described herein. The cells may bemixed with a cryoprotectant and stored or packaged into kits. Onceobtained, the stem cells and/or progenitor cells can be expanded inculture.

Where a mixed population of cells is used, the proportion of stem cellswithin the test population can vary. For example, the population cancontain few stem cells (e.g., about 1-10%) a moderate proportion of stemcells (e.g., about 10-90% (e.g., about 20, 25, 30, 40, 50, 60, 70, 75,80, or 85% stem cells)) or many stem cells (e.g., at least 90% of thepopulation (e.g., 92, 94, 96, 97, 98, or 99%) can be stem cells). Thecells will have the potential to differentiate into a completely orpartially differentiated cell of the inner ear (e.g., the cell can be apluripotent stem cell that differentiates into a cell that expresses oneor more auditory proteins). Partially differentiated cells are useful inthe treatment methods (whether therapeutic or prophylactic) so long asthey express a sufficient number and type of auditory-specific proteinsto confer a benefit on the patient (e.g., improved hearing).

Differentiation Methods

In general, differentiation can be promoted by contacting a suitabletarget cell and/or cell population with one or more of the compoundsdescribed herein for a time sufficient to promote complete or partialdifferentiation of the cells to or towards a mature sensory cell of theinner ear, e.g., a hair cell.

Suitable cells, e.g., identified according to the methods describedabove, can be cultured in vitro. In general, standard culture methodsare used in the methods described herein. Appropriate culture medium isdescribed in the art, such as in Li et al. Nature Medicine 9:1293-1299,2003. The growth medium for cultured stem cells can contain one or moreor any combination of growth factors. For example, growth media cancontain leukemia inhibitory factor (LIF), which prevents stem cells fromdifferentiating.

Cells can be separated into individual well of a culture dish andcultured. Formation of spheres (clonal floating colonies) from theisolated cells can be monitored, and the spheres can be amplified bydisrupting them (e.g., by physically means) to separate the cells, andthe cells can be cultured again to form additional spheres. Suchcultured cells can then be contacted with one or more of the compoundsdescribed herein.

Alternatively or in addition, cells may be contacted with one or more ofthe compounds described herein in combination with an additionalinduction protocol. There are a number of induction protocols known inthe art for inducing differentiation of stem cells with neurogenicpotential into neural progenitor cells, including growth factortreatment (e.g., treatment with EGF, FGF, and IGF, as described herein)and neurotrophin treatment (e.g., treatment with NT3 and BDNF, asdescribed herein). Other differentiation protocols are known in the art;see, e.g., Corrales et al., J. Neurobiol. 66(13):1489-500 (2006); Kim etal., Nature 418:50-6 (2002); Lee et al., Nat. Biotechnol. 18:675-9(2000); and Li et al., Nat. Biotechnol. 23:215-21 (2005).

As one example of an additional induction protocol, suitable cells aregrown in the presence of supplemental growth factors that inducedifferentiation into progenitor cells. These supplemental growth factorsare added to the culture medium. The type and concentration of thesupplemental growth factors is be adjusted to modulate the growthcharacteristics of the cells (e.g., to stimulate or sensitize the cellsto differentiate) and to permit the survival of the differentiated cellssuch as neurons, glial cells, supporting cells or hair cells.

Exemplary supplementary growth factors include, but are not limited tobasic fibroblast growth factor (bFGF), insulin-like growth factor (IGF),and epidermal growth factor (EGF). Alternatively, the supplementalgrowth factors can include the neurotrophic factors neurotrophin-3 (NT3)and brain derived neurotrophic factor (BDNF). Exemplary concentrationsof growth factors can range, e.g., from about 100 ng/mL to about 0.5ng/mL (e.g., from about 80 ng/mL to about 3 ng/mL, such as about 60ng/mL, about 50 ng/mL, about 40 ng/mL, about 30 ng/mL, about 20 ng/mL,about 10 ng/mL, or about 5 ng/mL).

Alternatively or in addition, the medium can be exchanged for mediumlacking growth factors. For example, the medium can be serum-freeDMEM/high glucose and F12 media (mixed 1:1) supplemented with N2 and B27solutions. Equivalent alternative media and nutrients can also be used.Culture conditions can be optimized using methods known in the art.

In some embodiments, a compound can be tested for its ability to promotedifferentiation using stem cells that have been engineered to express areporter gene that facilitates detection of cells converted into innerear cells. These engineered stem cells make up a reporter cell line. Areporter gene is any gene whose expression may be assayed; such genesinclude, without limitation, green fluorescent protein (GFP),α-glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT),horseradish peroxidase (HRP), alkaline phosphatase, acetylcholinesteraseand β-galactosidase. Other optional fluorescent reporter genes includebut are not limited to red fluorescent protein (RFP), cyan fluorescentprotein (CFP) and blue fluorescent protein (BFP), or any pairedcombination thereof, provided the paired proteins fluoresce atdistinguishable wavelengths.

A reporter gene can be under control of a promoter that is active incells of the inner ear, including progenitor cells and cells at varyingdegrees of differentiation, but not in stem cells. Ideally, the promoteris stably upregulated in the differentiated cells or progenitors cellsto allow assessment of the partially or fully differentiated phenotype(e.g., expression of the reporter gene and further identification ofgenes known to be expressed in the inner ear).

Methods for Analyzing Complete or Partial Differentiation

Cells that have been contacted with one or more of the compoundsdisclosed herein may be analyzed to determine if complete of partialdifferentiation has occurred. Such a determination can be performed byanalyzing the presence or absence of tissue specific genes, as describedabove (see Cell Selection). Alternatively or in addition, a hair cellcan be identified by physiological testing to determine if the cellsgenerate conductance channels characteristic of mature hair or spiralganglion cells. Such cells can be distinguished from spiral gangliacells using the markers described above.

Secondary assays can be used to confirm, or provide additional evidence,that a cell has differentiated into a cell of the inner ear. Forexample, a gene useful as a marker for identifying a cell of the innerear can be expressed exclusively in a particular cell type (e.g.,exclusively in a hair cell or exclusively in cells of the spiralganglion), or the cell may also be expressed in a few other cell types(preferably not more than one, two, three, four, or five other celltypes). For example, ephrinB1 and ephrinB2 are expressed in spiralganglion cells, and also in retinal cells. Thus detection of ephrinB1 orephrinB2 expression is not definitive proof that a stem cell hasdifferentiated into a cell of the spiral ganglion. Secondary assays canbe used to confirm that a cell has developed into a cell of the spiralganglion. Such assays include detection of multiple genes known to beexpressed in the suspected cell type. For example, a cell that expressesephrinB1 and/or ephrinB2, can also be assayed for expression of one ormore of GATA3, trkB, trkC, BF1, FGF10, FGF3, CSP, GFAP, and Islet1. Adetermination that these additional genes are expressed is additionalevidence that a stem cell has differentiated into a spiral ganglioncell.

Secondary assays also include detection of the absence of geneexpression or the absence of proteins that are not typically expressedin hair cells. Such negative markers include the pan-cytokeratin gene,which is not expressed in mature hair cells but is expressed insupporting cells of the inner ear (Li et al., Nat. Med. 9:1293-1299,2003).

Cells that are confirmed to have undergone complete or partialdifferentiation towards a inner ear sensory cell, e.g., a hair cell canbe transplanted or implanted into a patient.

Implantation Methods

Partially and/or fully differentiated cells, e.g., generated by themethods described above, can be transplanted or implanted, such as inthe form of a cell suspension, into the ear by injection, such as intothe luminae of the cochlea. Injection can be, for example, through theround window of the ear or through the bony capsule surrounding thecochlea. The cells can be injected through the round window into theauditory nerve trunk in the internal auditory meatus or into the scalatympani.

To improve the ability of transplanted or implanted cells to engraft,cells can be modified prior to differentiation. For example, the cellscan be engineered to overexpress one or more anti-apoptotic genes in theprogenitor or differentiated cells. The Fak tyrosine kinase or Akt genesare candidate anti-apoptotic genes that can be useful for this purpose;overexpression of FAK or Akt can prevent cell death in spiral ganglioncells and encourage engraftment when transplanted into another tissue,such as an explanted organ of Corti (see for example, Mangi et al., Nat.Med. 9:1195-201, 2003). Neural progenitor cells overexpressing α_(v)β₃integrin may have an enhanced ability to extend neurites into a tissueexplant, as the integrin has been shown to mediate neurite extensionfrom spiral ganglion neurons on laminin substrates (Aletsee et al.,Audiol. Neurootol. 6:57-65, 2001). In another example, ephrinB2 andephrinB3 expression can be altered, such as by silencing with RNAi oroverexpression with an exogenously expressed cDNA, to modify EphA4signaling events. Spiral ganglion neurons have been shown to be guidedby signals from EphA4 that are mediated by cell surface expression ofephrin-B2 and -B3 (Brors et al., J. Comp. Neurol. 462:90-100, 2003).Inactivation of this guidance signal may enhance the number of neuronsthat reach their target in an adult inner ear. Exogenous factors such asthe neurotrophins BDNF and NT3, and LIF can be added to tissuetransplants to enhance the extension of neurites and their growthtowards a target tissue in vivo and in ex vivo tissue cultures. Neuriteextension of sensory neurons can be enhanced by the addition ofneurotrophins (BDNF, NT3) and LIF (Gillespie et al., NeuroReport12:275-279, 2001).

In some embodiments, the cells described herein can be used in acochlear implant, for example, as described in Edge et al., (U.S.Publication No. 2007/0093878). A cochlear implant is an electronicdevice that is used to improve hearing in humans who have experiencedhearing loss, particularly severe to profound hearing loss. Thesedevices typically include an “external” and an “internal” part. Theexternal part includes a microphone, which can be placed behind the ear,that detects sounds in the environment. The sounds are then digitizedand processed by a small computer called a speech processor. Theexternal components may be referred to as a processor unit. In additionto the microphone and speech processor, the external portion of theimplant can include a power source, such as a battery and an externalantenna transmitter coil. The internal part is an electronic device thatis put under the skin in the vicinity of the ear and is commonlyreferred to as a stimulator/receiver unit (see FIG. 1). The coded signaloutput by the speech processor is transmitted transcutaneously to theimplanted stimulator/receiver unit situated within a recess of thetemporal bone of the implantee. This transcutaneous transmission occursthrough use of an inductive coupling provided between the externalantenna transmitter coil which is positioned to communicate with theimplanted antenna receiver coil provided with the stimulator/receiverunit. The communication is typically provided by a radio frequency (RF)link, but other such links have been proposed and implemented withvarying degrees of success.

The implanted stimulator/receiver unit typically includes the antennareceiver coil that receives the coded signal and power from the externalprocessor component, and a stimulator that processes the coded signaland outputs a stimulation signal to an electrode assembly, which appliesthe electrical stimulation directly to the auditory nerve producing ahearing sensation corresponding to the original detected sound.

An electrode connected to the electronic device is inserted into theinner ear. The electrode can be a bundle of wires that have opencontacts spread along the length of the cochlea and represent differentfrequencies of sounds. The number of electrodes can vary from 1 to about30 electrodes, such as about 5, 10, 15, 18, 20, 22, 24, 26, or 28electrodes.

Combination Therapies

In some embodiments, the present invention provides methods for treatinga patient with one or more of the compounds described herein using thedirect administration and cell therapy methods described above.

Routes of Administration for the Treatment of Abnormal CellProliferation

The route of administration will vary depending on the disease beingtreated. Abnormal cell proliferation and/or cancer can be treated usingdirect therapy, e.g., using systemic administration and/or localadministration according to one or more of the Food and DrugAdministration approved methods, for example, as described in CDER DataStandards Manual, version number 004 (which is available atfda.give/cder/dsm/DRG/drg00301.htm).

In some embodiments, the route of administration can be determined by apatient's health care provider or clinician, for example following anevaluation of the patient. In some embodiments, a individual patient'stherapy may be customized, e.g., one or more compounds, the routes ofadministration, and the frequency of administration can be personalized.Alternatively, therapy may be performed using a standard course oftreatment, e.g., using one or more pre-selected compounds andpre-selected routes of administration and frequency of administration.

In some embodiments, one or more of the compounds described herein canbe administered to a patient, e.g., a patient identified as being inneed of treatment for hair cell loss, using a systemic route ofadministration. Systemic routes of administration can include, but arenot limited to, parenteral routes of administration, e.g., intravenousinjection, intramuscular injection, and intraperitoneal injection;enteral routes of administration e.g., administration by the oral route,lozenges, compressed tablets, pills, tablets, capsules, drops, syrups,suspensions and emulsions; rectal administration, e.g., a rectalsuppository or enema; a vaginal suppository; a urethral suppository;transdermal routes of administration; and inhalation (e.g., nasalsprays).

Alternatively or in addition, one or more of the compounds describedherein can be administered to a patient, e.g., a patient identified asbeing in need of treatment for hair cell loss, using a local route ofadministration. For example, one or more of the compounds can beadministered during a surgical procedure, e.g., to remove a tumor andcan be performed by injection or topically at one or more site in andaround the cancerous site.

Pharmaceutical Formulations

Pharmaceutical compositions containing one or more of the compoundsdescribed herein (i.e., as active ingredients) will be formulatedaccording to the intended method of administration.

One or more of the compounds described herein can be formulated aspharmaceutical compositions for direct administration to a subject.Pharmaceutical compositions containing one or more of the compoundsdescribed herein can be formulated in a conventional manner using one ormore physiologically acceptable carriers or excipients. For example, apharmaceutical composition can be formulated for local or systemicadministration, e.g., administration by drops or injection into the ear,insufflation (such as into the ear), intravenous, topical, or oraladministration.

The nature of the pharmaceutical compositions for administration isdependent on the mode of administration and can readily be determined byone of ordinary skill in the art. In some embodiments, thepharmaceutical composition is sterile or sterilizable. The therapeuticcompositions featured in the invention can contain carriers orexcipients, many of which are known to skilled artisans. Excipients thatcan be used include buffers (for example, citrate buffer, phosphatebuffer, acetate buffer, and bicarbonate buffer), amino acids, urea,alcohols, ascorbic acid, phospholipids, polypeptides (for example, serumalbumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, water,and glycerol. The nucleic acids, polypeptides, small molecules, andother modulatory compounds featured in the invention can be administeredby any standard route of administration. For example, administration canbe parenteral, intravenous, subcutaneous, or oral. A modulatory compoundcan be formulated in various ways, according to the corresponding routeof administration. For example, liquid solutions can be made foradministration by drops into the ear, for injection, or for ingestion;gels or powders can be made for ingestion or topical application.Methods for making such formulations are well known and can be found in,for example, Remington's Pharmaceutical Sciences, 18th Ed., Gennaro,ed., Mack Publishing Co., Easton, Pa., 1990.

One or more of the compounds described herein can be administered, e.g.,as a pharmaceutical composition, directly and/or locally by injection orthrough surgical placement, e.g., to the inner ear and/or the colon. Theamount of the pharmaceutical composition may be described as theeffective amount or the amount of a cell-based composition may bedescribed as a therapeutically effective amount. Where application overa period of time is advisable or desirable, the compositions of theinvention can be placed in sustained released formulations orimplantable devices (e.g., a pump).

Alternatively or in addition, the pharmaceutical compositions can beformulated for systemic parenteral administration by injection, forexample, by bolus injection or continuous infusion. Such formulationscan be presented in unit dosage form, for example, in ampoules or inmulti-dose containers, with an added preservative. The compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, for example, sterile pyrogen-free water, before use.

In addition to the formulations described previously, the compositionscan also be formulated as a depot preparation. Such long actingformulations can be administered by implantation (e.g., subcutaneously).Thus, for example, the compositions can be formulated with suitablepolymeric or hydrophobic materials (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions formulated for systemic oral administrationcan take the form of tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents (forexample, pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (for example, lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(for example, magnesium stearate, talc or silica); disintegrants (forexample, potato starch or sodium starch glycolate); or wetting agents(for example, sodium lauryl sulphate). The tablets can be coated bymethods well known in the art. Liquid preparations for oraladministration may take the form of, for example, solutions, syrups orsuspensions, or they may be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations may be prepared by conventional means with pharmaceuticallyacceptable additives such as suspending agents (for example, sorbitolsyrup, cellulose derivatives or hydrogenated edible fats); emulsifyingagents (for example, lecithin or acacia); non-aqueous vehicles (forexample, almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (for example, methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration may be suitablyformulated to give controlled release of the active compound.

In some embodiments, the pharmaceutical compositions described hereincan include one or more of the compounds formulated according to any ofthe methods described above, and one or more cells obtained to themethods described herein.

Effective/Therapeutic Dose

Toxicity and therapeutic efficacy of the compounds and pharmaceuticalcompositions described herein can be determined by standardpharmaceutical procedures, using either cells in culture or experimentalanimals to determine the LD₅₀ (the dose lethal to 50% of the population)and the ED₅₀ (the dose therapeutically effective in 50% of thepopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index and can be expressed as the ratio LD₅₀/ED₅₀.Polypeptides or other compounds that exhibit large therapeutic indicesare preferred.

Data obtained from cell culture assays and further animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity, andwith little or no adverse effect on a human's ability to hear. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the methods described herein, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (that is, the concentrationof the test compound which achieves a half-maximal inhibition ofsymptoms) as determined in cell culture. Such information can be used tomore accurately determine useful doses in humans. Exemplary dosageamounts of a differentiation agent are at least from about 0.01 to 3000mg per day, e.g., at least about 0.00001, 0.0001, 0.001, 0.01, 0.1, 1,2, 5, 10, 25, 50, 100, 200, 500, 1000, 2000, or 3000 mg per kg per day,or more.

The formulations and routes of administration can be tailored to thedisease or disorder being treated, and for the specific human beingtreated. A subject can receive a dose of the agent once or twice or moredaily for one week, one month, six months, one year, or more. Thetreatment can continue indefinitely, such as throughout the lifetime ofthe human. Treatment can be administered at regular or irregularintervals (once every other day or twice per week), and the dosage andtiming of the administration can be adjusted throughout the course ofthe treatment. The dosage can remain constant over the course of thetreatment regimen, or it can be decreased or increased over the courseof the treatment.

Generally the dosage facilitates an intended purpose for bothprophylaxis and treatment without undesirable side effects, such astoxicity, irritation or allergic response. Although individual needs mayvary, the determination of optimal ranges for effective amounts offormulations is within the skill of the art. Human doses can readily beextrapolated from animal studies (Katocs et al., Chapter 27 inRemington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990). Generally, the dosage required toprovide an effective amount of a formulation, which can be adjusted byone skilled in the art, will vary depending on several factors,including the age, health, physical condition, weight, type and extentof the disease or disorder of the recipient, frequency of treatment, thenature of concurrent therapy, if required, and the nature and scope ofthe desired effect(s) (Nies et al., Chapter 3, In: Goodman & Gilman'sThe Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al.,eds., McGraw-Hill, New York, N.Y., 1996).

Kits

The compounds and pharmaceutical compositions described herein can beprovided in a kit, as can cells that have been induced to differentiate(e.g., stem cells, progenitor cells, and/or support cells that havedifferentiated into, for example, hair cells or hair-like cells) and/orthat are capable of differentiating into hair cells. The kit can alsoinclude combinations of the compounds and pharmaceutical compositionsdescribed herein and such cells. The kit can include (a) one or morecompounds, such as in a composition that includes the compound, (b)cells that have been induced to differentiate (e.g., stem cells,progenitor cells, and/or support cells that have differentiated into,for example, hair cells or hair-like cells) and/or that are capable ofdifferentiating into hair cells, (c) informational material, and anycombination of (a)-(c). In some embodiments, (a) and/or (b) can beprovided in a syringe (e.g., a preloaded disposable single dose syringe)suitable for the direct administration of (a) and/or (b) directly intothe ear (e.g., the middle or inner ear) of a patient. In someembodiments, (a) and/or (b) can be provided in a catheter and pumpsystem, as described above, suitable for the direct administration of(a) and/or (b) directly into the ear (e.g., the middle or inner ear) ofa patient. The informational material can be descriptive, instructional,marketing or other material that relates to the methods described hereinand/or to the use of the agent for the methods described herein. Forexample, the informational material relates to the use of the compoundto treat a subject who has, or who is at risk for developing, a auditoryhair cell loss hearing and/or abnormal cell proliferation. The kits canalso include paraphernalia for administering a differentiation agent toa cell (in culture or in vivo) and/or for administering a cell to apatient.

In one embodiment, the informational material can include instructionsfor administering the pharmaceutical composition and/or cell(s) in asuitable manner to treat a human, e.g., in a suitable dose, dosage form,or mode of administration (e.g., a dose, dosage form, or mode ofadministration described herein). In another embodiment, theinformational material can include instructions to administer thepharmaceutical composition to a suitable subject, e.g., a human, e.g., ahuman having, or at risk for developing, auditory hair cell loss and/orabnormal cell proliferation.

The informational material of the kits is not limited in its form. Inmany cases, the informational material (e.g., instructions) is providedin printed matter, such as in a printed text, drawing, and/orphotograph, such as a label or printed sheet. However, the informationalmaterial can also be provided in other formats, such as Braille,computer readable material, video recording, or audio recording. Ofcourse, the informational material can also be provided in anycombination of formats.

In addition to the differentiation agent, the composition of the kit caninclude other ingredients, such as a solvent or buffer, a stabilizer, apreservative, a fragrance or other cosmetic ingredient, and/or a secondagent for treating a condition or disorder described herein.Alternatively, the other ingredients can be included in the kit, but indifferent compositions or containers than the agent. In suchembodiments, the kit can include instructions for admixing the agent andthe other ingredients, or for using one or more compounds together withthe other ingredients.

The kit can include one or more containers for the pharmaceuticalcomposition. In some embodiments, the kit contains separate containers,dividers or compartments for the composition and informational material.For example, the composition can be contained in a bottle (e.g., adropper bottle, such as for administering drops into the ear), vial, orsyringe, and the informational material can be contained in a plasticsleeve or packet. In other embodiments, the separate elements of the kitare contained within a single, undivided container. For example, thecomposition is contained in a bottle, vial or syringe that has attachedthereto the informational material in the form of a label. In someembodiments, the kit includes a plurality (e.g., a pack) of individualcontainers, each containing one or more unit dosage forms (e.g., adosage form described herein) of the pharmaceutical composition. Forexample, the kit can include a plurality of syringes, ampoules, foilpackets, or blister packs, each containing a single unit dose of thepharmaceutical composition. The containers of the kits can be air tightand/or waterproof, and the containers can be labeled for a particularuse. For example, a container can be labeled for use to treat a hearingdisorder.

As noted above, the kits optionally include a device suitable foradministration of the composition (e.g., a syringe, pipette, forceps,dropper (e.g., ear dropper), swab (e.g., a cotton swab or wooden swab),or any such delivery device).

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 High Throughput Screen Optimization

A human embryonic kidney (HEK) cell line stably expressing a Luciferasegene controlled by an Math1 enhancer and minimal promoter was used in ahigh-throughput screen (HTS) of 144,000 small molecules to identifycompounds that increase Math1 expression (i.e., transcription and/ortranslation). Such compounds can be used to increase the conversion ofstem cells, progenitor cells, and support cells to or towards a haircell. Screens to identify such compounds are described by Li et al.,(U.S. Publication No. 2005/0287127) and Li et al., (U.S. applicationSer. No. 11/953,797).

The HTS was optimized using 1 μM retinoic acid as a positive control forthe activation of the Math1-luciferase reporter construct. Retinoic acidis a well known general inducer of differentiation to mature cell types.

HEK cells stably expressing the Math1 Luciferase reporter were platedonto 384 well plates and cultured overnight in medium containing 10%fetal calf serum (FCS). Activation of the Math1 enhancer was measured bythe increase in luminescence on a plate reader following addition of aluciferase detection agent. The assay was optimized for cell number,time of incubation, volume of medium, cell lysis reagent and luciferasereagent. Luminescence levels were compared in cells with theMath1-luciferase construct with and without retinoic acid (1 μM), and/ora luciferase construct with and without the Math1 enhancer and minimalpromoter region.

Data indicated that the Math1 luciferase reporter was sensitive toretinoic acid and that the assay had a low background. There wasconsistently a 1.8-fold increase in luciferase activity in cells treatedwith 1 μM retinoic acid compared to non-treated cells. Luminescencelevels from the promoter without the enhancer were low.

To improve assay sensitivity, and reduce the coefficient of variation(CV), a more sensitive luciferase reagent (BriteLite luciferase reporterassay reagent, Perkin Elmer) was used, and Triton-X-100 was added to thelysis reagent to ensure complete lysis of the cells. Following thesechanges a CV of 4.2% was recorded. These conditions were used for allhigh-throughput screens. The luminescence threshold for a compound to bepositive was defined as a 2-fold increase above the control (e.g., cellsexposed to DMSO).

Time of exposure to the compounds and cell density was optimized asfollows. The optimal exposure time was determined to provide the time atwhich Math1 activity was greatest with minimal cell loss. This wasperformed using various concentrations of retinoic acid.

Maximal luminescence was observed following a 60 hour incubation in thepresence of retinoic acid, however, a significant reduction of signal to50% was observed at the 72 hour time point. At the 48 hour time point,the luminescence was close to the plateau and has the highestsignal-to-background ratio of the times tested. 48 hours was, therefore,selected as the endpoint of the assay.

Optimal cell density was performed by performing a cell titrationexperiment in which the activity of the Math1 Luciferase reporter wascompared in wells seeded with 2000 to 40000 cells per well in a 384-wellplate. Cells exposed to retinoic acid were then compared to cells notexposed to retinoic acid. A cell viability assay was also performed toascertain the viability of the cells expressing the Math1 luciferasereporter.

The cell viability assay showed a linear increase in the number of cellsin the range between 2000 and 10000 cells per well. There was nodifference in the signal produced in the wells with 8000 and 15000 cellsper well, which implied that there may be reduced survival over thecourse of the experiment at densities greater than 10000 cells per well.The largest difference between untreated and retinoic acid treated cellswas observed at a density of 8000 cells per well. Based on theseresults, assay parameters were selected to be 8000 cells per well, witha 48 hour incubation period in the presence of absence of a testcompound or known activator.

Example 2 High Throughput Screening to Identify Activators of Atoh-1Expression

Cells were seeded on 384 well plates and allowed to attach overnight at37° C. with 5% CO2 in the absence of growth factors. Math1 activationwas measured by the increase in luminescence on a plate reader followingaddition of a luciferase detection reagent. Luminescence was assessed at24, 48, and 72 hours. These conditions were used to screen 144,000compounds contained in the small molecule libraries at HarvardUniversity's Laboratory for Drug Discovery in Neurodegeneration (LDDN).

HTS were performed using HEK-Math1 cells seeded in 384 well plates. Onecompound was added per well using pin transfer. The final concentrationof each compound was 100 μM. Cell were incubated in the presence of acompound for 48 hours at 37° C. with 5% CO2. Cell lysates then werecollected and bioluminescence determined. Luminescence values werecompared normalized against DMSO.

About 20000 compounds were screened per week in 50 plates of 384 wellwith the aid of robotic systems (Beckman Biomek FX). Compounds werescreened at an average final concentration of 0.7 μM (in 0.04% DMSO)with each plate containing 16 wells of 1 μM retinoic acid, as a positivecontrol, and 16 wells of 0.04% DMSO, as a vehicle control. Percentactivation of luminescence in the test compounds was determined forcells treated with the compounds against cells treated with DMSO.

Initial screens were performed using duplicate plates to assay a libraryof over 10,000 compounds. As shown in FIG. 117B, 40 compounds wereobserved in to increase Math1 enhancer activity by at least 2-fold,compared to the Math1 expression level observed for DMSO (i.e., 40compounds were positive). The Z factor for variation among wells withpositive control are shown in FIG. 117A. The Z factor is a statisticalparameter for HTS that reflects the quality of the data for each assayplate based on the magnitude of the signal window between the positiveand negative controls. The signal variability with the controls was thencalculated (Zhang et al., J. Biomol. Screen. 4:67-73, 1999). Any platewith a Z factor of less than 0.4 were repeated.

As shown in FIG. 118A and FIG. 118B, similar results were observed inrepeat duplicate experiments. This result the reliability andreproducibility of the HTS methods.

Of the 144,000 compounds screened, 921 were found to promote an increasein Math1 Luciferase reporter expression of greater than 60%. The hitrate was 0.47%. The maximum activation observed was 160% (e.g., comparedto DMSO).

Each of these compounds were then retested for dose-dependence ofresponse at final assay concentrations of 0.1, 1, and 5 μM. Among the921 positive compounds identified, 789 compounds evoked a increasingdoes response at 0.1, 1, and 10 μM concentrations. This observationsupports the specificity of the hits. In total, of the 921 compoundstested, 82% reproducibly activated the Math1 Luciferase reporter and 29%showed some toxicity.

Following these experiments, the compounds were re-evaluated and thosewith good physical chemical properties (i.e., low molecular weight, lackof reactive functional side groups or other undesirable molecularmotifs) that exhibited potent activation of Math1-luciferase and notoxicity were further investigated.

The total number of compounds selected for further evaluation is 110compounds. The structures of these compounds are shown in FIG. 1 to FIG.8. The increase in Math1 expression promoted by these compounds is shownin FIGS. 9-116.

Example 3 Evaluation of Positive Compounds by RT-PCR

Ten compounds, randomly selected from the group of 110 compoundsidentified in Example 2, were further evaluated, as follows. HEK cellswere plated on 96 well plates at a density of 100000 cells per well. Oneday after plating, 0.1, 1, and 5 μM of each positive compound was addedper well in a 10% fetal bovine serum (FBS) DMEM solution. Cells werethen lysed 48 hours after addition of the compound and Math1 expressionwas analyzed using a luciferase reporter assay, as described above, andreal-time PCR.

As shown in FIG. 119, four of the ten randomly selected compoundspromoted a greater than two fold increase in Math1 expression.

As shown in FIG. 120A, in agreement with the HTS, all four randomlyselected compounds promoted a greater than 2 fold increase in Math1expression as determined using the luciferase reporter assay.

RNA was also isolated and Math1 transcripts were amplified usinghigh-throughput RT-PCR. As shown in FIG. 120C, all four randomlyselected compounds promoted an increase in Math1 mRNA expression by atleast 2-fold compared to the DMSO control. CP.-0193184 and CP.-0000540promoted the highest increase in Math1 mRNA expression. FIG. 120B showsthe structures of the four randomly selected samples.

These data confirm the data presented in Example 2.

Example 4 Evaluation of Positive Compounds by Hair Cell Differentiation

Inner ear stem cells were exposed to compound CP.-0000540, which wasselected at random from the group of 110 positive compounds described inExample 2.

As shown in FIG. 121B, CP.-0000540 increased the number of cells thatco-labeled with hair cell specific markers Math1-GFP and myosin 7a whencompared to cells not exposed to compound CP.-0000540, as shown in FIG.121A. Hair cell differentiation was increased to 5.1% of total cellscompared to 1.6% for control.

Example 5 Stage Two Evaluation of Positive Compounds

All positive compounds were assessed for their ability to upregulateMath1 mRNA to confirm the observations made in Example 2.

Math1 mRNA expression levels were analyzed using RT-PCR as described inExample 3.

Example 6 Stage Three Evaluation of Positive Compounds

Positive compounds were assessed for their ability to increase the yieldof hair cells from mouse inner ear derived stem cells. Positivecompounds are also assessed in vivo in the inner ear of a sensorineuraldisease mouse model.

Isolated cells were exposed to 0.1, 1, and 5 μM of each positivecompound identified in Examples 3, 4, and 5 in vitro.

Compounds are also added to the inner ear of damaged inner ear mousemodels, e.g., gentamycin treated mouse models and/or fox caspasetransgenic mouse models. These models are used to test the compoundscapacity to regenerate hair cells after their loss by toxin damage, asoccurs in human deafness.

In vitro results were evaluated by detecting one or more of the haircell specific markers myosin VIIa, Math1, espin, Brn3.1, F-actin(phalloidin), α-9-acetylcholine receptor, and/or p27kip1. Antibodieswere incubate with cultured cells and detected by binding of secondaryantibodies coupled to FITC or rhodamine. The fluorescence in individualcells was viewed using confocal microscopy and the percentage ofpositive cells quantified. The effect of the three differentconcentrations of each compound was also determined.

Cells are also subjected to physiological testing to identify channelsthat would be present in mature hair cells were (1) present and (2)active.

In vivo results are assessed at 4, 8, and 12 week time points. Hair cellregeneration is assessed using immunocytochemistry, as described above.Functional recovery is assessed using methods performed routinely inspecialized suites for small animal physiology.

Example 7 Evaluation of Inner Ear Progenitor Cell Differentiation

A select number of positive compounds are assessed for their ability topromote differentiation of inner ear progenitor cells derived from bonemarrow to hair cells. Experiments are initially performed using bonemarrow derived inner ear progenitor cells (e.g., mesenchymal stem cells(MSCs)), and a luciferase reporter construct in which luciferaseexpression is driven by a myosin VIII enhancer region and promoter. Thisis a strong promoter in hair cells. The myosin VIIa enhancer region andpromoter was also operably linked to GFP. Positive results are confirmedusing RT-PCR and immunocytochemistry using the methods described inExample 3-6, modified for myosin VIIa.

Example 8 Pharmacological Characterization of the Compounds

The half maximal inhibitory concentration (IC₅₀) and median lethal doseor Lethal Dose, 50% (LD50) were determined for each of the compoundsidentified in Example 2 using standard laboratory techniques. IC₅₀ is ameasure of the effectiveness of a compound in inhibiting biological orbiochemical function. LD50 is the amount of a compound required to killhalf the members of a tested population.

IC₅₀ and LD50 values for each compound are shown in Table 2.

TABLE 2 Maximum LD50 Compound IC50 (μM) (Fold DMSO) (μM) CP-0000477 22 >30 CP-0000489 0.1 2.1 >30 CP-0000540 1.3 2 >30 CP-0000550 0.03 2 >30CP-0000553 0.01 2 >30 CP-0000554 0.4 2 >30 CP-0000557 0.02 2.6 30CP-0000571 0.3 2 >30 CP-0000928 0.3 2 >10 CP-0005186 1 1.8 >10CP-0007991 0.6 2 >10 CP-0007994 1 1.9 >10 CP-0008545 0.4 2.5 >10CP-0009883 2.5 2.3 >10 CP-0010539 0.5 2.1 >10 CP-0029278 0.5 1.9 >10CP-0029300 1.6 2 >10 CP-0034360 1 2 >10 CP-0036187 1 2.3 >10 CP-00390731.5 2.4 >10 CP-0045061 3 1.9 >10 CP-0047659 1.3 2.3 >10 CP-0050095 1.52.6 >10 CP-0059547 1 2 >10 CP-0059563 >10 1.9 >10 CP-0059642 1 2.2 >10CP-0060729 0.2 1.6 >10 CP-0060852 2.5 1.8 >10 CP-0061401 0.6 1.8 >10CP-0061566 0.2 2 >10 CP-0061777 >10 2.1 >10 CP-0062030 2 1.6 >10CP-0063182 0.2 1.7 >10 CP-0063375 2.2 1.9 >10 CP-0063508 >10 1.7 >10CP-0064231 1 1.5 >10 CP-0064314 >10 1.7 >10 CP-0064917 >10 2 >10CP-0065665 0.8 1.7 >10 CP-0066751 1 1.9 >10 CP-0066829 1 1.8 >10CP-0067108 3 1.8 >10 CP-0067233 2 1.8 >10 CP-0067246 1.8 1.5 >10CP-0068395 3 1.6 >10 CP-0068577 1 1.6 >10 CP-0068929 0.4 2 >10CP-0069934 2 1.7 >10 CP-0069961 2 1.7 >10 CP-0070164 1.6 1.7 >10CP-0070367 2 1.7 >10 CP-0070844 2 1.7 >10 CP-0070871 3 2 >10 CP-00708861 2.1 >10 CP-0071862 0.7 1.8 >10 CP-0072036 1.5 1.7 >10 CP-0072092 11.7 >10 CP-0072096 6 2.2 >10 CP-0072156 2 2 >10 CP-0072253 1.3 2 >10CP-0072271 1 2.2 >10 CP-0072720 3 1.8 >10 CP-0074806 0.7 1.8 >10CP-0075627 8 2 >10 CP-0076627 5 2.2 >10 CP-0078448 >10 1.9 >10CP-0079810 3 2.3 >10 CP-0079983 3 1.8 >10 CP-0080276 0.3 2 >10CP-0080773 >10 2.2 >10 CP-0087336 0.08 2.2 >10 CP-0087799 1 2.1 >10CP-0089966 >10 2.2 >10 CP-0091818 0.9 2.1 >10 CP-0096433 3 2 >10CP-0099289 >10 1.6 >10 CP-0102404 >10 1.6 >10 CP-0103978 1 2 >10CP-0104765 0.4 2 >10 CP-0104766 3 3 >10 CP-0104904 >10 2.2 >10CP-0105343 3 2 >10 CP-0105777 0.3 2 >10 CP-0107060 0.1 2.2 >10CP-0109953 0.1 1.8 >10 CP-0110352 0.05 1.8 >10 CP-0110644 1 1.8 >10CP-0130586 0.5 2.2 >10 CP-0130665 0.3 2 >10 CP-0131763 2 2.4 >10CP-0134381 2 2.2 >10 CP-0193184 >10 N/A >30

Example 9 Characterization of Compounds Using Inner Ear Progenitor CellsIsolated from Mouse Cochlea

Compounds identified by the methods described in Example 2 are testedfor their ability to promote the differentiation of mouse inner earprogenitor cells isolated from mouse cochlea to hair cells.

Cochlear stem cells are isolated from Atoh1-nGFP mice, as previouslydescribed (Oshima et al., supra). As described above, these animalsexpress a nuclear version of enhanced green fluorescent protein (GFP)when Atoh1 enhancer elements are activated (Chen et al. and Lumpkin etal., supra). Thus, cells obtained from these animals can be used totrack the differentiation of inner ear progenitor cells to hair cellsusing fluorescence microscopy.

Briefly, inner ear progenitor cells are obtained at 1 to 3 days of agefrom second or third generation animals. Cells are then seeded at adensity of 300 spheres per well (Oshima et al., J Assoc Res Otolaryngol8:18-31, 2007, and Martinez-Monedero et al., J Neurobiol 66:319-331,2008) and allowed to attached the surface of 6-well plates and culturedin the presence of growth factors. Cells cultured in DMEM mediumcontaining N2 and B27, but without growth factors, are exposed to thecompound and maintained in culture for 3-10 days. Cell differentiationis monitored by examining nuclear GFP expression green fluorescence fromthe Atoh1 reporter and by staining for the mature hair cell markersmyosin VIIa and espin in cultures treated with the compound as comparedto controls at 24, 72 m and 108 hour time points.

Positive results are confirmed using RT-PCR and immunocytochemistryusing the methods described in Examples 3-6 and 7.

Example 10 Characterization of Compounds Using Mouse Organ of CortiExplants

Compounds identified in Example 2 are tested for their ability topromote new hair cell formation in mouse organ of corti explants.

Briefly, explants are prepared from an Atoh1-GFP mouse by dissection.Organs of corti are cultured on collagen coated plates, and culturedovernight in serum containing medium. Compounds are added to thecultures at the time of plating, as previously described (Shi et al.,and Martinez-Monedero et al., supra). Cultures are maintained in DMEMcontaining B27 supplement (Invitrogen) for 3-10 days prior to analysis.

Hair cell formation arising from the epithelial cells in the culturesoutside of the hair cells rows is monitored using quantitativeimmunohistochemistry using an automated system to detect the appearanceof GFP-positive cells.

Example 11 Compound Optimization

Compounds are optimized to provide potency in the nanomolar range andreduced cytotoxicity.

Compounds are modified using the medicinal chemistry methods describedabove. Absorption, Distribution, Metabolism, and Excretion (ADME)studies are conducted to assess Log P determination, aqueous solubilityassessments, mouse liver microsomal stability determinations, and plasmaprotein binding analyses, as described below.

The following tests are performed using compounds synthesized andpurified to at least 95% as determined by 1H NMR. Additional analyticaltechniques (i.e. 13C NMR, IR, melting point, MS and/or elementalanalysis) are also used to determine structure and purity. Opticallypure materials are also assessed by chiral stationary-phase HPLC.Compound structures are assessed using 2-D NMR, and x-raycrystallography.

Compound Log P values are determined by adding 15 mL of compound stocksolution (10 mM in DMSO) to 750 mL 1-octanol buffer (pH 7.4) in testtubes. 3 mL testosterone (50 mM in DMSO) is used as a control. Thesamples are rotated at room temperature for 1 hour before allowingsamples to stand for 1 hour to allow separation of layers. 400 mL ofeach layer is removed and placed in separate containers. Serialdilutions of each sample in 50% aqueous methanol are then made. Standardcurves of compound and testosterone were prepared using 50% aqueousmethanol and samples are analyzed using LC/MS monitoring. The ratio ofcalculated concentration of test compound in each phase is calculatedindependently using the least dilute sample from each phase that fallswithin the standard curve for each of the two replicate experiments. LogP is calculated by taking the Log₁₀ of the average of the two calculatedratios.

Aqueous solubility assessments of the compounds is determined bycombining a minimum of 1 mg of each test compound with 1 mL of 0.07 MNaH₂PO₄ buffer solution adjusted to pH 7.4. Samples are then shaken for2 hours before being allowed to stand at room temperature for 12 hours.Samples are then filtered through a 0.45 micron nylon syringe filtersaturated with the sample. The resulting filtrate is assayed (N=2) byLC/MS using electrospray ionization.

Compound chemical metabolic stability is determined using pooled mouseliver microsomes. Compounds combined with 1 mg/mL microsomal protein and1 mM NADPH are incubated for 0, 15, 30 and 60 min. Testosterone andpropanolol are used as positive controls. Compound and microsomes in theabsence of NADPH are used as negative controls. Samples are quenchedwith acetonitrile and centrifuged for 10 min at 10,000 RPM toprecipitate proteins. Sample supernatants are analyzed (N=3) usingLC/MS. Standard curves are generated at four concentrations (100%, 30%,10% and 3%) and the remaining test compound remaining is determined atfour time points.

Plasma protein binding studies are performed by preparing solutionscontaining compound (5 μm, 0.5% final DMSO concentration), buffer and10% plasma (v/v in buffer). 96-well dialysis plates are assembled inwhich each well is divided in two by a semi-permeable cellulose membrane(molecular weight cut off 10,000). Buffer solutions are added to oneside of the membrane and the plasma solution to the other side. Theplates are then sealed and placed on an orbital shaker and incubated at37° C. Standards prepared in plasma and buffer are incubated at 37° C.with the dialysis plate. Corresponding solutions for each compound areanalyzed in cassettes by tandem mass spectrometry (LC-MS-MS). Eachcompound is tested in duplicate.

After equilibration, samples are taken from both sides of the membrane.Solutions for each batch of compounds are combined into two groups(plasma-free and plasma-containing) and cassette analyzed by LC-MS-MSusing two sets of calibration standards for plasma-free (6 points) andplasma-containing solutions (7 points). Samples are quantified todetermine the amount of compound bound using standard curves prepared inthe equivalent matrix.

Example 12 In Vivo Pharmacokinetic, Toxicity, and Formulation Studies

Compounds are administered using intracerebroventricular (icy) injectionto the mouse brain and dosing studies are performed to determine themaximal dose of the compound that can be administered via this route.

Pharmacokinetic studies are performed using intraperitoneal (IP) orintra-cochlear administration of 3 mg/kg compound to determine dosingand the concentration of the compound in the cochlea, the relevanttissue. Compound levels are measured in the plasma and cochlear tissueat nine time points spanning 24 hours.

Orally administered compounds are dissolved in a formulation such as 2%hydroxypropyl-beta-cyclodextrin at a concentration of 3 mg/kg bodyweight. Intra-cochlear administered compounds are administered aspreviously described (Chen et al., J. Neurosci. Methods, 150:67-73,2006). Briefly, mice are anesthetized and a tube is inserted into acochleostomy to provide access to the scala tympani. Compounds insolution are then delivered by a syringe pump at a flow rate of 1 μL perhour over a 6 hour time period. Surgical sites are then closed and theanimals are tested at various time points.

Example 13: In Vivo Studies Using Animal Deafness Model

A 10-wk old mouse is exposed to octave-band (8-16 kHz) noise at ˜116 dBSPL for 2 hours. In CBA/CaJ mice, this noise dose destroys the outerhair cells throughout the basal half of the cochlea and inner hair cellsand supporting cells are destroyed in a more restricted region in themiddle of the cochlea (Wang et al., J. Ass. Res. Otolaryngol.,3:248-268, 2002). Further, neurons begin to degenerate within 7 days inthe regions in which inner hair cells are destroyed. Such mice aretested for ABRs and DPOAEs after a recovery period. Cochleas weredissected and subjected to immunostaining after cutting of frozensections or whole mounts. Cell division is assessed in the animals byinjecting BrdU and antibody staining frozen sections. Cell death isevaluated by TUNEL. The number of hair cells, supporting cells andspiral ganglion neurons were counted.

Noise treated mice are injected with compounds (1 mg/10 g body weight)one week after noise treatment. Samples are analyzed at 4, 8, 14, and 21day time points and hair cell counts are performed for the entire lengthof the cochlear spiral.

Functional assessment is performed using measurements of amplitudeversus level functions for DPOAE and ABR, as previously described(Kujawa and Liberman, J. Neurophysiol, 78:3095-3106, 1997; and Maison etal., J. Neurophysiol, 90:2941-2949, 2003).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of treating hearing impairment orimbalance disorder in a subject in need thereof, comprising selectingthe subject in need of treatment, and administering to the subject atherapeutically effective amount of a pharmaceutical compositioncomprising a compound having the formula:

wherein, X is S; R₅₀ is hydrogen; R₅₃, is —C(O)R^(q); R₅₁ and R₅₂ areeach, independently, hydrogen; R^(q) is NR^(s)R^(t) wherein one of R^(s)and R^(t) is hydrogen, and the other is: (a) C₆-C₁₀ aryl or heteroarylincluding 5-10 atoms, each of which is optionally substituted with from1-5 R^(r); (b) C₁-C₆ alkyl, which is substituted with phenoxy that isoptionally substituted with from 1-5 R^(r); or (c) —O—N═C(NH₂)(C₆-C₁₀aryl), wherein the aryl portion is optionally substituted with from 1-5R^(r); and R^(r) at each occurrence is, independently: (i) halo; NH₂;NH(C₁-C₃ alkyl); N(C₁-C₃ alkyl)₂; hydroxy; C₁-C₆ alkoxy or C₁-C₆haloalkoxy; nitro; or cyano; or (ii) C₁-C₆ alkyl or C₁-C₆ haloalkyl; ora pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the hearing impairment or imbalance disorder is associated withloss of auditory hair cells in the subject.
 3. The method of claim 1,and wherein the pharmaceutical composition is administered locally tothe inner ear of the subject.
 4. The method of claim 1, wherein thepharmaceutical composition increases the number of cells in thesubject's inner ear that have characteristics of auditory hair cells. 5.The method of claim 1 wherein the pharmaceutical composition comprises acompound having the formula:

or a pharmaceutically acceptable salt thereof.
 6. The method of claim 1wherein the pharmaceutical composition comprises a compound having theformula:

or a pharmaceutically acceptable salt thereof.
 7. The method of claim 3,wherein the pharmaceutical composition is administered by application tothe luminae of the cochlea, to the auditory nerve trunk in the internalauditory meatus, to the scala tympani, or if present, into a cochlearimplant.
 8. The method of claim 1, wherein the hearing impairment orimbalance disorder is an auditory disorder resulting from a loss of haircells resulting in sensorineural hearing loss, deafness or a vestibulardisorder.